EA040196B1 - AML ANTIGENS AND THEIR APPLICATIONS - Google Patents

Description

The present invention relates to the field of biology, immunology and medicine.

Acute myeloid leukemia (AML) is a high-risk cancer with a five-year survival rate of 40-50% in patients younger than 60 years of age. For patients older than 65 years, survival rates are even lower, with only less than 20% of patients experiencing sustained remissions. In the treatment of acute leukemia, allogeneic stem cell transplantation is often used. This method was originally developed to avoid exposing patients to otherwise lethal myeloablative chemotherapy, but has since been found to cause complications associated with an alloreactive immune response (graft-versus-host disease, GVHD). Decreasing the number (depletion) of T cells in grafts before reinfusion prevented GVHD, but transplant recipients with T cell depletion, as well as transplant recipients from their own identical twin donor, had a much higher recurrence rate, which is evident suggests that the success of allogeneic stem cell transplantation (TSC) depends on the induction of an immune response against leukemia (graft response against leukemia (GTL)). This has led to the development of strategies for using allogeneic stem cell transplantation that does not use myeloablative conditions (Reduced Intensity Stem Cell Transplantation, RIST) to reduce cytotoxicity and perform allogeneic SCT in a larger group of patients, including elderly patients and patients previously treated with intensive treatment. Preparatory treatment regimens for RIST aim to suppress the acquired immune system of the recipients to prevent graft rejection without complete removal of the bone marrow in the recipients, thus reducing the early toxicity of TSCs. After transplantation, the recipient's stem cells are gradually replaced by the donor's stem cells, and complete donor chimerism is usually achieved within three months after TSC. Although allogeneic TSC leads to the recovery of a significant number of patients, and great progress has been made in the maintenance therapy of TSC recipients, still 15 to 30% of patients die as a result of complications associated with transplantation, such as GVHD, and infectious diseases. complications (arising as a result of slow recovery of immunity after TSC or as a complication as a result of immunosuppressive therapy for GVHD). Therefore, although TSC leads to effective recovery when strong graft-against-leukemia (GTL) responses are induced, its therapeutic success is limited by anti-host immune responses leading to GVHD, which causes high morbidity and mortality. In view of the high incidence of GVHD after allogeneic stem cell transplantation, which is fatal in 15-30% of patients, and the fact that a suitable donor is not always available for a given patient, alternative treatments would be preferred. International patent application PCT/NL 2014/050873 proposes patient-derived AML-specific human antibodies that are capable of binding intact AML cells. It is important to note that the antibodies are obtained from AML patients, which are people who have undergone allogeneic TSC and are in complete remission, indicating that these antibodies are effective against AML cells. Therefore, the use of the antibodies described in PCT/NL 2014/050873 in AML therapy is preferred.

Also, instead of passive immunization with antibodies, the use of immunotherapy may be preferable. In immunotherapy, a patient suffering from a disease is provided with a disease-specific antigen that induces and/or enhances an immune response in said patient against the disease. Also preferred may be prophylactic or semi-prophylactic applications in which the individual is provided with a disease-specific antigen to elicit an immune response prior to the onset of development or before (after) the progression of the disease. For example, immunization using an AML-specific target molecule to elicit an immune response against AML would be particularly advantageous for patients undergoing allogeneic hematopoietic stem cell transplantation. Another group of patients for whom immunization using such a target would be preferable are patients suffering from medium to high risk myelodysplastic syndrome (MDS). Such patients have a moderate to high risk of developing AML, so it is desirable to elicit an immune response against AML in advance. The risk that a patient's MDS will progress to AML is usually rated according to the International Prognosis Scoring Scale (IPSS, see e.g. Malcovati et al., 2013). Non-limiting examples of patients with moderate to high risk MDS are patients with myelodysplastic syndrome with refractory anemia with an excess of type I and II blasts (MDS-RAEB-1 and MDS-RAEB-2).

Immunotherapy and vaccinations that are specifically directed against AML are not currently available due to the lack of suitable AML-specific antigens.

AML-specific antigens will also be particularly suitable for determining whether a patient sample contains antibodies and/or immune cells capable of specifically binding AML cells. Such information, for example, would be valuable for diagnosing AML or for tracking the progress of AML therapy.

The aim of the present invention is to provide new peptides and compounds containing

- 1 040196 AML cell antigen. In a preferred embodiment, peptides and compounds are provided that are capable of eliciting and/or eliciting an immune response, preferably a specific immune response against myeloproliferative disorders, more preferably AML.

The present invention provides an isolated recombinant or purified CD43 peptide with a length of not more than 100 amino acid residues, while the specified peptide contains an amino acid sequence of at least 3 amino acid residues and not more than 51 amino acid residues, which is identical to the sequence located between positions 133 and 184 of the amino acid sequence CD43 as shown in Fig. 13. Said peptide preferably contains an amino acid sequence of at least 3 amino acid residues and no more than 51 amino acid residues that is identical to the sequence located between positions 133 and 183 of the CD43 amino acid sequence as shown in FIG. 13. In some embodiments, the length of said amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues, or at least amino acid residues, or at least 13 amino acid residues, or at least amino acid residues, or at least 15 amino acid residues, or at least amino acid residues, or at least 25 amino acid residues, or at least amino acid residues, or at least 35 amino acid residues residues, or at least amino acid residues, or at least 45 amino acid residues, or at least amino acid residues, or 51 amino acid residues.

In some embodiments, an isolated recombinant or purified CD43 peptide of no more than 100 amino acid residues is provided, wherein said peptide contains an amino acid sequence of at least 3 amino acid residues and no more than 33 amino acid residues that is identical to the sequence located between amino acid positions 133 and 165 CD43 sequences as shown in FIG. 13. In some embodiments, the length of said amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 25 amino acid residues, or at least 30 amino acid residues, or 33 amino acid residue.

In some embodiments, an isolated recombinant or purified CD43 peptide of no more than 100 amino acid residues is provided, wherein said peptide contains an amino acid sequence of at least 3 amino acid residues and no more than 15 amino acid residues that is identical to the sequence located between positions 133 and 147 of the sequence. amino acids CD43 as shown in FIG. 13. In some embodiments, the length of said amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or 15 amino acid residues.

The present inventors have surprisingly found that a specific immune response against AML can be detected and/or elicited using the CD43 peptide of the present invention. This finding was unexpected because CD43 is present on the surface of most types of (non-cancerous) leukocytes, as well as on the surface of many non-hematopoietic tumor cells, such as human colon cancer cells, human cervical cancer cells, human lung cancer cells, and breast adenocarcinoma cells. human glands. Given this wide distribution of the CD43 peptide, prior to the invention, CD43 was not considered as a suitable compound to provide AML specificity. However, as shown in the examples, the AML-specific antibody AT14-013 binds the CD43 peptide of the present invention. In addition, the AT14-013 antibody binds to a variety of different CD43+ AML cells (FIGS. 2 and 3), but does not bind to CD43+ peripheral blood mononuclear cells (PBMC), activated and inactivated T cells, B cells, inactivated monocytes, thymocytes, ALL cells, colon carcinoma cells, non-malignant colon cells, Jurkat cells, Ramos cells, or normal bone marrow cells (shown in FIG. 5). Therefore, the AT14-013 antibody, which is specifically directed against the CD43 peptide as defined in the claims, binds CD43+ AML cells, while it does not bind various other types of CD43+ cells. Interestingly, the AT14-013 antibody does not bind various types of non-AML CD43+ hematopoietic stem cells or more mature lymphoid lineage cells. The examples also show that the AT14-013 antibody is able to bind fetal hematopoietic stem cells, which concludes that the CD43 epitope that is recognized by AT14-013 is an oncofetal epitope. The AT14-013 antibody can also bind autologous leukemic stem cells. Moreover, the AT14-013 antibody is able to counteract the growth of AML cells in vivo. Thus, the present invention also provides the CD43 antigen of AML cells.

CD43, also referred to as leukosialin, sialophorin, galactoglycoprotein, leukocyte sialoglycoprotein, or gp115, is a type I glycosylated mucin-like transmembrane protein that is present on the surface of most hematopoietic cells, with the exception of erythrocytes. CD43, encoded by a single exon, plays a role in intercellular interactions. It contains a highly glycosylated extracellular region of 235 amino acids. Two glycoforms of CD43 have been described, with one glycoform primarily containing tetrasaccharides and the other glycoform containing primarily branched hexasaccharides. Both glycoforms can be expressed on the surface of the same cell. CD43, for example, is described in Shelley et al. (1989) and Schmid et al. (1992). The human CD43 sequence shown in FIG. 13 is listed in the Genbank CCDS database as CCDS10650.1.

In the context of the present description, the term CD43 peptide according to the present invention refers to a chain of amino acids with a length of not more than 100 amino acid residues, while the specified amino acid chain contains a sequence of at least 3 amino acid residues and not more than 51 amino acid residues, which is identical to the sequence located between 133 and 184 amino acid sequence positions of the human CD43 protein as shown in FIG. 13, or wherein said amino acid chain contains a sequence of at least 3 amino acid residues and not more than 51 amino acid residues that is identical to the sequence located between amino acid positions 133 and 183 of the human CD43 protein amino acid sequence as shown in FIG. 13, or wherein said amino acid chain contains a sequence of at least 3 amino acid residues and not more than 33 amino acid residues, which is identical to the sequence located between amino acid positions 133 and 165 of the amino acid sequence of the human CD43 protein, as shown in FIG. 13, or wherein said amino acid chain contains a sequence of at least 3 amino acid residues and no more than 15 amino acid residues that is identical to the sequence located between amino acid positions 133 and 147 of the amino acid sequence of the human CD43 protein as shown in FIG. 13.

As explained in detail in the examples, the present invention provides insight that the amino acid sequence of the human CD43 protein between positions 133 and 184 contains an AML epitope to which the AT14-013 antibody specifically binds. Said AML epitope contains at least one amino acid residue that is present between positions 133 and 165 of the amino acid sequence, as shown in FIG. 13. Said AML epitope, which is present on the surface of various AML cell lines and AML blasts and which is absent or not exposed on the surface of many other CD43+ cells, is therefore particularly suitable for eliciting or detecting an AML-specific immune response. In some embodiments, the CD43 peptide of the present invention comprises the amino acid sequence GTITTNSPETSSSRTS. In some embodiments, the CD43 peptide of the present invention comprises the amino acid sequence GTITTNSPETSSRTSGAPVTTAASSLETSRGTS.

In some embodiments, the CD43 peptide of the present invention comprises the amino acid sequence GTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVSLETSKGTSG.

In some embodiments, the length of the CD43 peptide according to the present invention is at least 90 amino acid residues. In some embodiments, the CD43 peptide of the present invention is no more than 85 amino acid residues, or no more than 75 amino acid residues, or no more than 70 amino acid residues. In some embodiments, the CD43 peptide of the present invention is no more than 65 amino acid residues, or no more than 60 amino acid residues, or no more than 55 amino acid residues, or no more than 50 amino acid residues, or no more than 45 amino acid residues, or no more than 40 amino acid residues, or not more than 35 amino acid residues. In some embodiments, said CD43 peptide of the present invention is no more than 52 amino acid residues, or no more than 51 amino acid residues, or no more than 33 amino acid residues, or no more than 30 amino acid residues, or no more than 25 amino acid residues, or no more than 20 amino acid residues, or not more than 15 amino acid residues. In some embodiments, the CD43 peptide of the present invention is at least 5 amino acid residues in length, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues.

In some embodiments, said CD43 peptide of the present invention is at least 52 amino acid residues long, or at least 51 amino acid residues long, wherein said peptide contains an amino acid sequence that is identical to a sequence located between positions 133 and 184 of the amino acid sequence. human CD43 protein as shown in FIG. 13. In some embodiments, said CD43 peptide of the present invention is at least 51 amino acid residues in length, wherein said amino acid residues are identical to amino acids located at positions 133,184 of the amino acid sequence of the human CD43 protein from 133 to 183, as shown in FIG. 13.

In some embodiments, the CD43 peptide of the present invention is at least 33 amino acid residues long and contains an amino acid sequence that is identical to the sequence between amino acid positions 133 and 165 of the human CD43 protein amino acid sequence as shown in FIG. 13.

In some embodiments, the CD43 peptide of the present invention is at least 15 amino acid residues long and contains an amino acid sequence that is identical to the sequence located between amino acid positions 133 and 147 of the human CD43 protein amino acid sequence as shown in FIG. 13.

In some embodiments, the CD43 peptide of the present invention consists of the sequence GTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPLTMATVSLETSKGTSG.

In some embodiments, the CD43 peptide of the present invention consists of the sequence GTITTNSPETSSRTSGAPVTTAASSLETSRGTS.

In some embodiments, the CD43 peptide of the present invention consists of the sequence GTITTNSPETSSSRTS.

In the context of the present description of the expression, the sequence located between the X and Y positions of the CD43 amino acid sequence, as shown in FIG. 13, the sequence located between the X and Y positions of the amino acid sequence of the human CD43 protein as shown in FIG. 13, wherein said amino acid residues are identical to the amino acids located between the X-Y positions of the amino acid sequence of the human CD43 protein as shown in FIG. 13 and an amino acid sequence that is identical to the sequence located between the X and Y positions of the amino acid sequence of the human CD43 protein as shown in FIG. 13 includes sequences that are located between the indicated positions and include the amino acid(s) at the X and/or Y positions. In addition, the terms include sequences that are located between the indicated positions and that do not contain the amino acid(s) at the X and/or Y positions. In other words, in some embodiments, the amino acid(s) at said X and/or Y positions are present in the CD43 peptide of the present invention, while in other embodiments, the amino acids at said X and/or Y positions are not present.

In addition to the indicated amino acid sequences that are identical to the sequence located between 133 and 184 positions of the amino acid sequence, or the sequence located between 133 and 183 positions of the amino acid sequence, or the sequence located between 133 and 165 positions of the amino acid sequence, or the sequence located between 133 and 147 amino acid sequence positions of the human CD43 protein as shown in FIG. 13, the CD43 peptide of the present invention may further contain other amino acid residues. In some embodiments, these other amino acid residues are not derived from a CD43 sequence. These other amino acid residues, which are also referred to here as non-CD43 amino acid residues, may, for example, function to improve stability and/or improve immunogenicity, and/or to attach the CD43 peptide to another moiety, such as, for example, a molecular scaffold or carrier. . Non-limiting examples of such scaffolds or carriers are Megathura crenulata limpet hemocyanin and CLIPS scaffolds (such as, for example, bis(bromomethyl)benzene, tris(bromomethyl)benzene and tetra(bromomethyl)benzene described in WO 2004/077062). Thus, in some embodiments, an isolated recombinant or purified peptide of no more than 100 amino acid residues is provided, wherein said peptide contains an amino acid sequence of at least 3 amino acid residues and no more than 52 amino acid residues, or no more than 51 amino acid residues that is identical to sequence located between positions 133 and 184 of the CD43 amino acid sequence as shown in FIG. 13, and wherein said peptide also contains at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80 amino acid residues other than CD43, wherein the full-length sequence of said amino acid residues other than from CD43 is absent in human CD43, as shown in FIG. 13.

In some embodiments, an isolated recombinant or purified peptide of no more than 100 amino acid residues is provided, wherein said peptide contains an amino acid sequence of at least 3 amino acid residues and no more than 51 amino acids.

- 4 040196 a lot residue which is identical to the sequence located between positions 133 and 183 of the CD43 amino acid sequence as shown in FIG. 13, and wherein said peptide also contains at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80 non-CD43 amino acid residues, wherein the full-length sequence of said non-CD43 amino acid residues residues are absent in human CD43, as shown in FIG. 13. In some embodiments, the length of said amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 25 amino acid residues, or at least 30 amino acid residues, or at least 35 amino acid residues, or at least 40 amino acid residues, or at least 45 amino acid residues, or at least 50 amino acid residues, or 51 amino acid residues.

In some embodiments, an isolated recombinant or purified peptide of no more than 100 amino acid residues is provided, wherein said peptide contains an amino acid sequence of at least 3 amino acid residues and no more than 33 amino acid residues that is identical to the sequence located between positions 133 and 165 of the amino acid sequence. CD43 as shown in Fig. 13, and wherein said peptide also contains at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80 non-CD43 amino acid residues, wherein the full-length sequence of said non-CD43 amino acid residues residues are absent in human CD43, as shown in FIG. 13. In some embodiments, the length of said amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 25 amino acid residues, or at least 30 amino acid residues, or 33 amino acid residue.

In some embodiments, an isolated recombinant or purified peptide of no more than 100 amino acid residues is provided, wherein said peptide contains an amino acid sequence of at least 3 amino acid residues and no more than 15 amino acid residues that is identical to the sequence located between positions 133 and 147 of the amino acid sequence. CD43 as shown in Fig. 13, and wherein said peptide also contains at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80 non-CD43 amino acid residues, wherein the full-length sequence of said non-CD43 amino acid residues residues are absent in human CD43, as shown in FIG. 13. In some embodiments, the length of said amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or 15 amino acid residues.

The term CD43 peptide according to the present invention also includes the aforementioned peptides.

In some embodiments, there is provided a compound comprising the CD43 peptide of the present invention. In some embodiments, an immunogenic compound comprising a CD43 peptide of the present invention is provided. In some embodiments, said CD43 peptide is attached to a pharmaceutically acceptable carrier or scaffold.

In some embodiments, the CD43 peptide of the present invention is a truncated CD43 molecule no longer than 100 amino acid residues. In a preferred embodiment, said truncated CD43 molecule lacks an intracellular region of wild-type human CD43 protein. In preferred embodiments, the truncated CD43 molecule lacks both the intracellular region and the transmembrane region of wild-type human CD43 protein. In other preferred embodiments, said CD43 peptide of the present invention is a truncated CD43 extracellular region of no more than 90 amino acid residues, or no more than 80 amino acid residues, or no more than 70 amino acid residues,

- 5 040196 or not more than 60 amino acid residues, or not more than 52 amino acid residues, or not more than 51 amino acid residues, or not more than 50 amino acid residues, or not more than 45 amino acid residues, or not more than 40 amino acid residues, or not more than 35 amino acid residues residues, or not more than 33 amino acid residues, or not more than 30 amino acid residues, or not more than 25 amino acid residues, or not more than 20 amino acid residues, which contains an amino acid sequence of at least 3 amino acid residues in length and not more than 52 amino acid residues or not more than 51 amino acid residues, which is identical to the sequence located between positions 133 and 184 of the CD43 amino acid sequence as shown in FIG. 13. In some embodiments, the length of said amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 25 amino acid residues, or at least 30 amino acid residues, or at least 35 amino acid residues, or at least 40 amino acid residues, or at least 45 amino acid residues, or at least 50 amino acid residues, or 51 amino acid residues.

In some embodiments, said CD43 peptide of the present invention is a truncated CD43 extracellular region of no more than 90 amino acid residues, or no more than 80 amino acid residues, or no more than 70 amino acid residues, or no more than 60 amino acid residues, or no more than 51 amino acid residues , or not more than 50 amino acid residues, or not more than 45 amino acid residues, or not more than 40 amino acid residues, or not more than 35 amino acid residues, or not more than 33 amino acid residues, or not more than 30 amino acid residues, or not more than 25 amino acid residues, or no more than 20 amino acid residues, which contains an amino acid sequence of at least 3 amino acid residues and no more than 51 amino acid residues, which is identical to the sequence located between positions 133 and 183 of the CD43 amino acid sequence as shown in FIG. 13. In some embodiments, the length of said amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 25 amino acid residues, or at least 30 amino acid residues, or at least 35 amino acid residues, or at least 40 amino acid residues, or at least 45 amino acid residues, or at least 50 amino acid residues, or 51 amino acid residues.

In some embodiments, said CD43 peptide of the present invention is a truncated CD43 extracellular region of at most 90 amino acid residues, or at most 80 amino acid residues, or at most 70 amino acid residues, or at most 60 amino acid residues, or at most 51 amino acid residues , or not more than 50 amino acid residues, or not more than 45 amino acid residues, or not more than 40 amino acid residues, or not more than 35 amino acid residues, or not more than 33 amino acid residues, or not more than 30 amino acid residues, or not more than 25 amino acid residues, or no more than 20 amino acid residues, which contains an amino acid sequence of at least 3 amino acid residues and no more than 33 amino acid residues, which is identical to the sequence located between positions 133 and 165 of the CD43 amino acid sequence as shown in FIG. 13. In some embodiments, the length of said amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 25 amino acid residues, or at least 30 amino acid residues, or 33 amino acid residue.

In some embodiments, said CD43 peptide of the present invention is a truncated CD43 extracellular region of no more than 90 amino acid residues, or no more than 80 amino acid residues, or no more than 70 amino acid residues, or no more than 60 amino acid residues, or no more than 51 amino acid residues , or not more than 50 amino acid residues, or not more than 45 amino acid residues, or not more than 40 amino acid residues, or not more than 35 amino acid residues, or not more than 33 amino acid residues, or not more than 30 amino acid residues, or not more than 25 amino acid residues, or not more than 20 amino acid residues, which contains an amino acid sequence with a length of at least 3 amino acid residues and not more than 15 amino acid residues, which is identical to the sequence located

- 6 040196 lying between positions 133 and 147 of the amino acid sequence of CD43, as shown in FIG. 13. In some embodiments, the length of said amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or 15 amino acid residues.

In some embodiments, a CD43 peptide of the present invention is provided which is a truncated CD43 extracellular region of no more than 90 amino acid residues, or no more than 80 amino acid residues, or no more than 70 amino acid residues, or no more than 60 amino acid residues, or no more than 52 amino acid residues, or not more than 51 amino acid residues, or not more than 50 amino acid residues, or not more than 45 amino acid residues, or not more than 40 amino acid residues, or not more than 35 amino acid residues, or not more than 33 amino acid residues, or not more than 30 amino acid residues, or not more than 25 amino acid residues, or not more than 20 amino acid residues, or not more than 15 amino acid residues.

As the person skilled in the art will know, once an immunogenic sequence has been proposed, it has become possible to vary the sequence to some extent, thereby preferably optimizing the immunogenicity and/or stability of the resulting immunogen. This is, for example, carried out by means of mutagenesis procedures, after which, in the preferred embodiment, the stability and/or immunogenicity of the compounds obtained are examined and an improved AML-specific antigenic compound is selected. One skilled in the art is able to generate variants of an antigen starting at a specific amino acid sequence. For example, a conservative amino acid substitution is used. Examples of conservative amino acid substitutions include replacing one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, with another hydrophobic residue, and replacing one polar residue with another polar residue, such as replacing arginine with lysine, glutamic acid with aspartic acid, or glutamine for asparagine. In some embodiments, a general substitution assay is performed, which includes replacing at least one amino acid residue with any other amino acid residue and examining the resulting compounds.

Thus, in some embodiments, the present invention provides an isolated recombinant or purified CD43 peptide of no more than 100 amino acid residues that contains or essentially consists of an amino acid sequence of at least 3 amino acid residues and no more than 52 amino acid residues or no more 51 amino acid residues that has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97 %, more preferably at least 98%, more preferably at least 99% sequence identity to a sequence located between amino acid positions 133 and 184 of the human CD43 protein sequence as shown in FIG. 13. In some embodiments, an isolated, recombinant, or purified CD43 peptide is provided that contains a 51 amino acid residue long amino acid sequence that has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to a sequence located between positions 133 and 183 of the amino acid sequence human CD43 protein as shown in FIG. 13.

In some embodiments, an isolated recombinant or purified CD43 peptide of no more than 100 amino acid residues is provided that contains or essentially consists of a 33 amino acid residue long amino acid sequence that has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to sequence located between positions 133 and 165 of the amino acid sequence of the human CD43 protein, as shown in FIG. 13.

In some embodiments, an isolated recombinant or purified CD43 peptide of no more than 100 amino acid residues is provided that contains or essentially consists of an amino acid sequence of 15 amino acid residues that has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to sequence located between positions 133 and 147 of the amino acid sequence of the human CD43 protein, as shown in FIG. 13.

The term % Sequence Identity is defined herein as the percentage of residues in a candidate amino acid sequence that are identical to residues in a reference sequence after two sequences have been aligned and gaps introduced as necessary to achieve maximum percent identity. Methods and computer programs for alignment are well known in the art. One computer program that can be used or adapted to determine if a candidate sequence falls under this definition is Align 2, made by Genentech, Inc., which has been filed with user documentation with the United States Copyright Office, Washington. , DC 20559, December 10, 1991

The isolated recombinant or purified CD43 peptide as defined herein is also referred to as the CD43 peptide of the present invention or the CD43 antigen of the present invention. In some embodiments, the amino acid residues of the CD43 peptide of the present invention are selected from 20 amino acid residues that occur naturally in eukaryotes, which are also referred to as standard or canonical amino acids. Alternatively, the CD43 peptide of the present invention contains non-natural amino acid residues such as, for example, D-amino acids (ie, D-stereoisomers of amino acids) or N-methyl amino acids.

The CD43 peptide of the present invention is preferably glycosylated. Such a peptide more accurately reflects the native AML antigen in vivo given the fact that the native CD43 protein on the cell surface is highly glycosylated. In some preferred embodiments, said CD43 peptide of the present invention contains sialic acid (also referred to as α-acetylneuraminic acid) residues. In vivo, human CD43 is highly sialylated. Treatment with neuraminidase cleaves sialic acid residues in the CD43 glycoprotein. Therefore, in some embodiments, the CD43 peptide of the present invention is neuraminidase sensitive. In some embodiments, a CD43 peptide of the present invention is provided that is oncosialylated, meaning that said CD43 peptide has a tumor-specific sialylation profile. In the context of the present description, a CD43 peptide of the present invention with a tumor-specific sialylation profile includes a CD43 peptide of the present invention with a sialylation profile that forms a tumor cell, or a CD43 peptide of the present invention with a sialylation profile that is either at least 90% identical, or at least 95%, or at least 97% similar to the sialylation profile that a tumor cell produces.

In some embodiments, a CD43 peptide of the present invention is provided that is produced by an AML cell. In some embodiments, a CD43 peptide of the present invention is provided that is produced by a cell line derived from an AML cell. With such a CD43 peptide, the glycosylation profile will more closely resemble the glycosylation profile of AML cells in an AML patient in vivo. In some embodiments, said CD43 peptide of the present invention is produced by a human acute monocytic leukemia (THP-1) cell. In some embodiments, said CD43 peptide of the present invention is produced by a Kasumi 3 cell, or an HL60 cell, or a KG1a cell, or an SH2 cell, or a MonoMac6 cell, or a Molm13 cell, or a CML K562 cell.

In the context of the present description, a CD43 peptide according to the present invention having a glycosylation profile similar or identical to the glycosylation profile that results from the expression of said CD43 peptide in an AML cell, such as an AML blast or an AML cell line, is referred to as a CD43 peptide according to the present invention. invention with an AML-specific glycosylation profile. Thus, in some embodiments, a CD43 peptide of the present invention is provided with an AML-specific glycosylation profile.

In some embodiments, a CD43 peptide of the present invention is provided that is produced by an MDS cell. In some embodiments, a CD43 peptide of the present invention is provided that is produced by a cell line derived from an MDS cell. With such a CD43 peptide, the glycosylation profile will more closely resemble the glycosylation profile of MDS cells in an in vivo MDS patient.

In some embodiments, CD43 peptides of the present invention are provided with an MDS-specific glycosylation profile. For such peptides, the glycosylation profile is similar or identical to the glycosylation profile that results from the expression of the CD43 peptide of the present invention in an MDS cell, eg an MDS blast or an MDS cell line.

In other embodiments, a CD43 peptide of the present invention is provided that is produced by a host cell using in vitro glycoengineered technology (eg, according to Roche Diagnostics GmbH). In these embodiments, the host cells provide

- 8 040196 enzymes a-2,6-sialyltransferase and/or a-2,3-sialyltransferase, so that upon receipt of the CD43 peptide according to the present invention, this peptide will be sialylated. Thus, a CD43 peptide according to the present invention is also provided, which is produced by a cell containing α-2,6-sialyltransferase and/or α-2,3-sialyltransferase. In some embodiments, said α-2,6-sialyltransferase and/or α-2,3-sialyltransferase includes exogenous α2,6-sialyltransferase and/or exogenous α-2,3-sialyltransferase, which means that said enzyme is recombinantly introduced into host cell (or to the parent host cell from which the current host cell is derived). In some embodiments, a CD43 peptide of the present invention is provided that is produced by a host cell containing an exogenous nucleic acid sequence encoding an α-2,6-sialyltransferase and/or an α-2,3-sialyltransferase.

Antibodies against CD43 are known in the art. However, it is clear that these antibodies recognize a different epitope. For example, in table. 1 in Kim et al., 2014 shows that anti-CD43 monoclonal antibodies (mAb) YG5, 2C8, 8E10 and DFT-1 do indeed bind AML cells, however, some or all of these known antibodies (Kim et al., 2014) also many other non-AML cells also bind, including CEM7, Jurkat, IM9, Ramos, Raji, Daudi, Reh, normal (healthy) bone marrow, and PBL cells. Therefore, the YG5, 2C8, 8E10 and DFT-1 antibodies are not entirely specific for AML. Conversely, the AT14-013 antibody, which specifically binds the CD43 peptides of the present invention, does not bind Jurkat cells, Ramos cells, normal (healthy) bone marrow cells, or PBL/PBMC cells (FIGS. 5 and 9B). Thus, the YG5, 2C8, 8E10 and DFT-1 antibodies bind another CD43 epitope.

The UNI antibody (Tuccillo et al., 2014a and Tuccillo et al., 2014b) recognizes a CD43 epitope, including a GalNac-O-linked monosaccharide corresponding to the Tn antigen of O-glycans. It can be concluded that the protein core of this epitope contains amino acids 64 to 83 of the CD43 amino acid sequence. This antigen is expressed in human thymocytes, HPB-ALL, H9, and Molt-4 leukemic cell lines, and in the peripheral blood CD4+ T-lymphocyte subpopulation. However, the AT14-013 antibody does not bind human thymocytes, indicating that the present invention provides a different AML antigen.

International patent application WO 2007/146172 describes antibodies 5F1, 51-41 and 138-10 that recognize CD43 present on the surface of the human colorectal adenocarcinoma cell line Colo205 and the human gastric carcinoma cell line NCI-N87. AML is not mentioned in WO 2007/146172. The AT14-013 antibody, which specifically binds the AML-specific CD43 peptides of the present invention, does not bind Colo205 cells as shown in the examples. Thus, antibodies 5F1, 51-41 and 138-10 bind another CD43 epitope.

International patent application WO 2006/121240 describes antibodies EB-1, EB-2 and EB-3 that are able to recognize the non-glycosylated region of CD43, consisting of amino acids at position 73-81 of the CD43 amino acid sequence. This antigen is present on the surface of thymocytes, some hematopoietic precursors in the bone marrow, on the surface of AML cells, acute lymphoblastic leukemia (ALL) cells and chronic myeloid leukemia (CML) cells. EB-1 recognizes its antigen in both sialidase-treated and untreated CD43 molecules. In contrast, the glycosylated CD43 peptides of the present invention are no longer recognized by the AT14-013 antibody after treatment with sialidase (neuramidase), which means that the present invention provides a neuramidase-sensitive AML antigen, unlike the antigens EB-1, EB-2 and EB- 3. In addition, the AT14-013 antibody does not bind ALL cells or thymocytes, unlike the EB-1, EB-2 and EB3 antibodies. In addition, the CD43 peptides of the present invention contain an amino acid sequence of at least 3 amino acid residues and not more than 51 amino acid residues, which is identical to the sequence located between amino acid residues 133 and 184 in the CD43 amino acid sequence, the domain of which is different from the domain formed by the amino acid residues at positions 73-81 of the CD43 amino acid sequence, which is recognized by the EB-1, EB-2 and EB-3 antibodies. Antibodies EB-1, EB-2 and EB-3 thus also bind another CD43 epitope. In conclusion, the present invention proposes a new AML-specific antigen.

The length of the CD43 peptide according to the present invention is preferably not more than 100 amino acid residues and at least 3 amino acid residues, preferably at least 5 amino acid residues, or at least 6 amino acid residues, or at least 7 amino acid residues, or at least at least 8 amino acid residues, or at least 9 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues. In some embodiments, the specified length is at least 20 amino acid residues. In some embodiments, the specified length is at least 25 amino acid residues. In some embodiments, the specified length is at least 30 amino acid residues. In some implementations

- 9 040196 the specified length is at least 33 amino acid residues. In some embodiments, the specified length is at least 35 amino acid residues. In some embodiments, the specified length is at least 40 amino acid residues. In some embodiments, the specified length is at least 45 amino acid residues. In some embodiments, the specified length is at least 50 amino acids. In some embodiments, the specified length is at least 51 amino acids. In some embodiments, the specified length is at least 52 amino acids.

In some embodiments, the specified length is not more than 90 amino acid residues, or not more than 85 amino acid residues, or not more than 75 amino acid residues, or not more than 70 amino acid residues, or not more than 65 amino acid residues, or not more than 60 amino acid residues, or not more than more than 55 amino acid residues, or not more than 52 amino acid residues, or not more than 51 amino acid residues, or not more than 50 amino acid residues, or not more than 45 amino acid residues, or not more than 40 amino acid residues, or not more than 35 amino acid residues, or at most 30 amino acid residues, or not more than 25 amino acid residues, or not more than 20 amino acids. In some embodiments, the length of said peptide is at most 52 amino acid residues and at least 3 amino acid residues, preferably at least 5 amino acid residues, or at least 6 amino acid residues, or at least 7 amino acid residues, or at least at least 8 amino acid residues, or at least 9 amino acid residues, or at least 10 amino acid residues. In some embodiments, the peptide is at most 51 amino acid residues and at least 3 amino acid residues, preferably at least 5 amino acid residues, or at least 6 amino acid residues, or at least 7 amino acid residues, or at least at least 8 amino acid residues, or at least 9 amino acid residues, or at least 10 amino acid residues. In some embodiments, the length of the specified peptide is not more than 33 amino acid residues and at least 3 amino acid residues, in the preferred embodiment, at least 5 amino acid residues, or at least 6 amino acid residues, or at least 7 amino acid residues, or at least at least 8 amino acid residues, or at least 9 amino acid residues, or at least 10 amino acid residues. In some embodiments, the length of said peptide is at most 15 amino acid residues and at least 3 amino acid residues, preferably at least 5 amino acid residues, or at least 6 amino acid residues, or at least 7 amino acid residues, or at least at least 8 amino acid residues, or at least 9 amino acid residues, or at least 10 amino acid residues.

In some embodiments, a CD43 peptide of the present invention consists of an amino acid sequence of at least 3 amino acid residues and no more than 51 amino acid residues that is identical to the sequence located between positions 133 and 183 of the CD43 amino acid sequence as shown in FIG. 13. In some embodiments, the CD43 peptide of the present invention consists of an amino acid sequence of at least 5 amino acid residues and no more than 40 amino acid residues that is identical to the sequence located between positions 133 and 183 of the CD43 amino acid sequence as shown in FIG. 13. In some embodiments, the CD43 peptide of the present invention consists of an amino acid sequence of at least 5 amino acid residues and no more than 33 amino acid residues that is identical to the sequence located between positions 133 and 183 of the CD43 amino acid sequence as shown in FIG. 13. In some embodiments, a CD43 peptide of the present invention consists of an amino acid sequence of at least 5 amino acid residues and no more than 20 amino acid residues that is identical to the sequence located between positions 133 and 183 of the CD43 amino acid sequence as shown in FIG. 13.

In some embodiments, the CD43 peptide of the present invention consists of an amino acid sequence of at least 5 amino acid residues and no more than 33 amino acid residues that is identical to the sequence located between positions 133 and 165 of the CD43 amino acid sequence as shown in FIG. 13. In some embodiments, a CD43 peptide of the present invention consists of an amino acid sequence of at least 5 amino acid residues and no more than 30 amino acid residues that is identical to the sequence located between positions 133 and 165 of the CD43 amino acid sequence as shown in FIG. 13. In some embodiments, a CD43 peptide of the present invention consists of an amino acid sequence of at least 5 amino acid residues and no more than 20 amino acid residues that is identical to the sequence located between positions 133 and 165 of the CD43 amino acid sequence as shown in FIG. 13.

In some embodiments, the CD43 peptide of the present invention consists of

- 10 040196 amino acid sequence of at least 5 amino acid residues and not more than 15 amino acid residues in length, which is identical to the sequence located between positions 133 and 147 of the CD43 amino acid sequence as shown in FIG. 13. In some embodiments, a CD43 peptide of the present invention consists of an amino acid sequence of at least 8 amino acid residues and no more than 15 amino acid residues that is identical to the sequence located between positions 133 and 147 of the CD43 amino acid sequence as shown in FIG. 13. In some embodiments, a CD43 peptide of the present invention consists of an amino acid sequence of at least 10 amino acid residues and no more than 15 amino acid residues that is identical to the sequence located between positions 133 and 147 of the CD43 amino acid sequence as shown in FIG. 13.

In some embodiments, the aforementioned peptides are glycosylated, preferably containing sialic acid residues to better mimic the natural AML-specific antigen. In some embodiments, the aforementioned peptides are oncosialylated. In some embodiments, the aforementioned peptides are produced by AML cells or AML cell lines, preferably THP-1 cells. In some embodiments, the aforementioned peptides are produced by MDS cells or an MDS cell line.

Nucleic acid molecules or their functional equivalents encoding the CD43 peptide according to the present invention are also covered by the present invention. In addition, for example, an isolated synthetic or recombinant nucleic acid molecule or a functional equivalent thereof encoding a CD43 peptide according to the present invention is also provided. In the context of the present description, the nucleic acid molecule or nucleic acid sequence according to the present invention preferably contains a chain of nucleotides, more preferably DNA, cDNA or RNA. In other embodiments, the nucleic acid molecule or nucleic acid sequence of the present invention comprises other kinds of nucleic acid structures such as, for example, a DNA/RNA helix, a peptide nucleic acid (PNA), a blocked nucleic acid (LNA) and/or a ribozyme. Such other nucleic acid structures are referred to as functional nucleic acid sequence equivalents. Thus, the term functional equivalent of a nucleic acid molecule includes a strand containing non-natural nucleotides, modified nucleotides, and/or non-nucleotide building blocks that perform the same function as naturally occurring nucleotides.

In some embodiments, a nucleic acid molecule or a functional equivalent thereof according to the present invention is provided, wherein the human nucleic acid sequence is codon optimized for a non-human cell, e.g., a non-human producer cell such as E. coli cells, cells Chinese hamster ovary (CHO), NSO cell (which is mouse myeloma), or 293 (T) cell. This means that at least one codon from said human nucleic acid sequence is replaced with at least one codon that is preferred by said non-human cell.

In the context of the present description, an isolated synthetic or recombinant nucleic acid molecule or a functional equivalent thereof encoding a CD43 peptide according to the present invention is also referred to as a nucleic acid molecule or a functional equivalent according to the present invention.

Now that the present invention provides CD43 peptides containing a new AML-specific antigen, many applications are possible. For example, in some embodiments, the CD43 peptide of the present invention is used to induce, isolate, and/or produce immune cells and/or antibodies, or functional portions or functional derivatives thereof, that are capable of specifically binding to lymphoproliferative and/or myeloproliferative cells. Immune cells and/or antibodies, or functional portions or functional derivatives thereof, which are induced, isolated and/or produced using the CD43 peptide of the present invention are particularly suitable for the treatment or prevention of myeloproliferative or lymphoproliferative disorders. Moreover, it should be taken into account that the AT14-013 antibody, which is an antibody specific for the CD43 peptide of the present invention, also targets leukemia stem cells, which are known to be more resistant to the effects of therapy and are often responsible for the manifestation recurrence of the disease after treatment. In some embodiments, the CD43 peptide of the present invention is used to induce and/or generate AML-specific immune cells and/or AML-specific antibodies. For example, a non-human animal is immunized with at least one CD43 peptide of the present invention, or an immunogenic compound comprising a CD43 peptide of the present invention, or a nucleic acid molecule or functional equivalent thereof encoding a CD43 peptide of the present invention.

- 11 040196 tenyu, or a vector containing a nucleic acid molecule or a functional equivalent according to the present invention, in the preferred embodiment, followed by at least one repeated (booster) introduction. Then, immune cells and/or antibodies that are specific for lymphoproliferative and/or myeloproliferative cells, preferably specific for AML, are collected from said non-human animal. In some embodiments, said immune cells comprise T cells, such as, for example, natural killer (NK) cells or T helpers.

In some embodiments, said immune cells harvested from said immunized non-human animal contain B cells, for example, AML-specific B cells are particularly suitable for generating AML-specific antibodies. AML-specific B cells collected from said immunized animal are used, for example, to obtain hybridomas from which AML-specific antibodies are obtained. In other embodiments, B cells harvested from said immunized animal are transduced with Bcl-6 and Bcl-xL nucleic acids and cultured in long-term ex vivo B cell cultures, such as described in EP 1974017 and US Pat. No. 91227251. In this way, long-term replicating cultures of B cells are obtained in which B cells both replicate and produce antibody. In some embodiments, AML-specific antibodies produced by said hybridomas or such B cell culture are harvested and, for example, used in anti-AML therapy, preferably after antibody humanization to reduce side effects. In some embodiments, an antibody and/or a B cell derived from said non-human animal is tested for competition with an AT14-013 antibody for binding to CD43. This is for example done by incubating AML cells with said antibody or a B cell derived from said non-human animal and then adding the AT14-013 antibody. As a control, AML cells are preferably incubated with AT14-013 antibody in the absence of any other antibody or B cell. If preincubation of AML cells with an antibody or B cell derived from said non-human animal appears to affect the binding of AT14-013 to said AML cells, then it is concluded that said antibody or B cell derived from said non-human animal competes with the AT14-013 antibody for binding to CD43.

In some embodiments, nucleic acid sequences encoding variable regions of B cells derived from said non-human animal are sequenced to obtain AML-specific variable domain nucleic acid sequences, after which at least one nucleic acid molecule containing such sequences is introduced into producer cells such as, for example, E. coli cells, Chinese Hamster Ovary (CHO) cells, NSO cells, or 293(T) cells to produce AML-specific antibodies. Said at least one nucleic acid sequence is preferably codon optimized for said producer cell. In the context of the present description, the term codon means a triplet of nucleotides (or their functional equivalents) that encode a specific amino acid residue. The term codon-optimized means that at least one codon from the animal's original nucleic acid sequence is replaced with at least one codon that is preferred by a cell derived from another species, such as, for example, a particular producer cell. These substitution codons preferably code for the same amino acid residue as the original codon being replaced in the animal.

In some embodiments, CD43-specific antibodies derived from said non-human animal or from immune cells of said non-human animal are humanized, meaning that at least a portion of the animal's amino acid sequence, preferably at least some or all of the framework sequence is replaced with a human sequence to reduce adverse side effects in humans.

Animal immunization protocols, including suitable administration procedures and adjuvants, methods for producing and purifying antibodies and/or immune cells derived from such immunized animals, competition experiments, and procedures for humanizing non-human antibodies are well known in the art. For example, references are provided in Hartley et al., 1995.

In some embodiments, a CD43 peptide of the present invention or a compound comprising a CD43 peptide of the present invention is used to screen a phage display library for the identification and/or isolation of AML-specific immunoglobulins (usually Fab fragments). In some embodiments, a naive phage display library is used. In preferred embodiments, a phage display library derived from at least one AML patient is used such that said library will already be targeted for AML. In some embodiments, an AML-specific immunoglobulin derived from said phage display library is tested for competition with the AT14-013 antibody for binding to CD43. This is, for example, done by applying a competition test, described

- 12 040196 sledge in this document.

Thus, the use of a CD43 peptide according to the present invention, or the use of an immunogenic compound according to the present invention, or the use of a nucleic acid molecule or a functional equivalent thereof encoding a CD43 peptide according to the present invention, or the use of a vector containing a nucleic acid molecule or a functional equivalent according to the present invention are also provided. of the invention, for inducing, isolating and/or obtaining an immune cell or an antibody or a functional part or functional equivalent thereof, such as, for example, a Fab fragment. The specified immune cell or antibody or its functional part or functional equivalent in the preferred embodiment, can specifically bind lymphoproliferative cells and/or myeloproliferative cells. Preferably, said myeloproliferative cells are AML cells, MDS cells and/or CML cells, most preferably AML cells. In some embodiments, a CD43 peptide of the present invention, or an immunogenic compound of the present invention, or a nucleic acid molecule or a functional equivalent of the present invention, or a vector containing a nucleic acid molecule or a functional equivalent of the present invention, is used to induce and/or generate an antibody. , which can induce antibody-mediated cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). A non-limiting example of such an antibody is AT14-013 as shown in the examples. The fact that a CD43 peptide of the present invention or an immunogenic compound of the present invention can induce and/or produce an antibody with ADCC and/or CDC-inducing activity means that antibodies can be induced or made to be functional in vivo. .

Also provided herein is a CD43 peptide or a compound of the present invention for use as an immunogen, as well as a nucleic acid molecule or a functional equivalent of the present invention or a vector containing a nucleic acid molecule or a functional equivalent of the present invention for use as an immunogen.

In some embodiments, a method is provided for producing immune cells and/or antibodies that can specifically bind lymphoproliferative cells and/or myeloproliferative cells, such as, for example, an AML-specific immune cell or an AML-specific antibody, said method comprising immunizing an animal, not which is a human, a CD43 peptide according to the present invention, or a compound according to the present invention, or a nucleic acid molecule, or a functional equivalent according to the present invention, or a vector containing a nucleic acid molecule or a functional equivalent according to the present invention. Said method preferably also includes selecting an immune cell and/or an antibody that can specifically bind lymphoproliferative cells and/or myeloproliferative cells from said non-human animal. In some embodiments, the AML-specific immune cell and/or AML-specific antibody is collected from said non-human animal. In some embodiments, a B cell and/or an antibody derived from said non-human animal is tested for competition with the AT14-013 antibody for binding to CD43. Also provided herein is an immune cell and/or antibody that can specifically bind lymphoproliferative cells and/or myeloproliferative cells obtainable by the method of the present invention. In some embodiments, there is provided an AML-specific antibody or an AML-specific immune cell obtainable by the method of the present invention for producing an AML-specific immune cell or an AML-specific antibody. Such an AML-specific antibody preferably competes with the AT14-013 antibody for binding to CD43.

Said non-human animal preferably includes a mammal such as a rodent or cattle. In some embodiments, said non-human animal includes a mouse, rat, rabbit, llama, camel, pig, poultry, cow, goat, horse, monkey, and/or gorilla.

Because the AT14-013 antibody specifically binds the CD43 peptide of the present invention, other antibodies that are made, produced, or selected using the CD43 peptide of the present invention will typically compete with the AT14-013 antibody for binding to CD43. Conversely, existing anti-CD43 antibodies that are known in the art do not compete with the AT14-013 antibody, while known such antibodies do compete with each other, as shown in FIG. 9. Thus, these other antibodies known in the art clearly bind an epitope that is different from that of AT14-013. Thus, an isolated recombinant or purified antibody, or a functional portion or functional equivalent thereof, is also provided that competes with the AT14-013 antibody for binding to CD43. Said isolated, recombinant, or purified antibody, or a functional portion or functional equivalent thereof, preferably competes with the antibody.

- 13 040196

AT14-013 for binding to at least a portion of an epitope that is located between amino acids 133 and 184 in the CD43 amino acid sequence as shown in FIG. 13. Said isolated recombinant or purified antibody, or a functional portion or functional equivalent thereof, typically competes with the AT14-013 antibody for binding to the CD43 peptide of the present invention.

In the context of the present description, the term antibody refers to an immunoglobulin protein containing two heavy chains linked to each other, with each heavy chain also connected to a light chain.

In the present description, the term functional part of an antibody is defined as a part that has at least one of the same general property as the specified antibody, but not necessarily in quantitative terms. Said functional moiety may bind the same antigen as said antibody, although not necessarily to the same extent. The functional portion of the antibody preferably contains at least a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the functional portion of an antibody comprises at least a heavy chain variable domain (VH). Non-limiting examples of a functional portion of an antibody are a single domain antibody, a single chain antibody, a nanobody, a unibody, a variable single chain fragment (scFv), a T-cell-attracting bispecific activator (BiTE), a Fab fragment, and an F(ab') ₂ fragment.

As used herein, the term antibody functional equivalent is defined as an artificial binding compound containing at least one CDR sequence of an antibody, preferably a heavy chain CDR3 sequence. Said functional equivalent preferably comprises the antibody heavy chain CDR3 sequence as well as the antibody light chain CDR3 sequence. More preferably, said functional equivalent comprises the heavy chain CDR1, CDR2, and CDR3 sequences of the antibody, and the light chain CDR1, CDR2, and CDR3 sequences of said antibody. The functional equivalent of an antibody is obtained, for example, by modifying the antibody such that at least the antigen-binding property of the resulting compound remains substantially the same, but not necessarily quantitatively. This is done in many ways, for example, by conservative amino acid substitution, and thereby the amino acid residue is replaced by another residue with usually similar properties (size, hydrophobicity, etc.), so that the full functioning of the specified antibody is essentially not impaired. A non-limiting example of a functional equivalent of an antibody is an antibody with a modified Fc tail, which, for example, is modified with amino acid substitution(s) and/or alteration(s) using glycosylation.

As is well known to one of skill in the art, an antibody heavy chain is the larger of the two kinds of chains that make up an immunoglobulin molecule. The heavy chain contains a constant domain and a variable domain, and this variable domain is involved in antigen binding. The light chain of an antibody is the smaller of the two kinds of chains that make up an immunoglobulin molecule. The light chain contains a constant domain and a variable domain. This variable domain is often, but not always, involved with the heavy chain variable domain in antigen binding.

Complementarity determining regions (CDRs) are hypervariable regions present in heavy chain variable domains and light chain variable domains. In the case of full antibodies, the heavy chain CDR1-3 and the adjacent light chain CDR1-3 together form the antigen binding site.

As used herein, an immune cell, antibody or functional portion or functional equivalent thereof is specific for AML if it is able to bind AML cells with a binding affinity that is at least twice that of the irrelevant one, which is control of the antibody or immune cell with said AML cells (wherein the control antibody or immune cell is not specific for said AML cells). For example, binding of AML cells to an AML-specific antibody, B cell, or T cell is typically mediated through the complementarity regions of the antibody, B cell receptor (BCR), or T cell receptor (TCR), respectively. The specific three-dimensional structure of both the AML antigen and the antibody variable domain or BCR or TCR allows the two structures to bind together (lock and key interaction), as opposed to random, non-specific attachment of antibodies or BCR or TCR. However, the term AML-specific includes some reactivity towards other cell types. Since antibodies or BCRs or TCRs usually recognize an epitope of an antigen, and the epitope as such may also be present on the surface of other cells, AML-specific antibodies, B cells or T cells can then recognize such other cells. Non-limiting examples of such other cells are MDS and CML cells. In addition, melanoma cells and melanocytes appear to be bound by the AML-specific antibody AT14-013, although to a lesser extent. Therefore, the term AML-specific does not exclude binding of antibodies or B cells or T cells to another cell that contains at least a portion of the same epitope. However, the examples show that many CD43+ cells, including CD43+ hematopoietic cells such as, for example, PBMCs, T cells and B cells, do not contain the AML antigen of the present invention.

The CD43 peptides of the present invention are particularly suitable for testing the presence of AML-specific binding compounds, such as, for example, AML-specific antibodies or AML-specific immune cells, such as B cells or T cells, in a biological sample. For example, a sample obtained from an individual, or a portion of such a sample that contains antibodies, B cells and/or T cells, is incubated with a CD43 peptide of the present invention or with a compound that contains a CD43 peptide of the present invention to screen for the presence of AML. -specific antibodies and/or AML-specific immune cells. In the event that such antibodies or immune cells are present in the specified sample or in the specified sample fraction and bind the specified CD43 peptide according to the present invention, the specified sample is typed as positive for AML-specific binding compounds (i.e. antibodies and/or immune cells ). Said sample, for example, includes a blood sample or a bone marrow or biopsy sample, such as, for example, myeloid sarcoma (which is also called chloroma).

An AML-specific antibody or an AML-specific immune cell, for example, is detected and/or quantified using an immunological assay such as, for example, Western blot, enzyme-captured immunosorbent assay (ELISA), or radioimmunoassay (RIA). Such research methods are well known in the art. The labeled CD43 peptides of the present invention are, for example, incubated with a blood sample or a bone marrow or biopsy sample, such as, for example, myeloid sarcoma, or with a fraction of such a sample that contains antibodies, B cells and/or T cells, after which unbound binding compounds are washed out. It is then determined whether AML-specific antibodies or immune cells bind to the CD43 peptides of the present invention. In some embodiments, an unlabeled CD43 peptide of the present invention or an unlabeled compound comprising a CD43 peptide of the present invention is contacted with a sample that contains antibodies and/or immune cells, such as, for example, a blood sample or a bone marrow sample, or a biopsy, such as, for example, myeloid sarcoma, or with a fraction of such a sample that contains antibodies, B cells and/or T cells. After incubation, at least one washing step is preferably carried out to remove unbound antibodies and unbound immune cells. It is then examined whether antibodies or immune cells have bound to said CD43 peptide according to the present invention, for example, using an antibody that is specifically directed against human antibodies or human immune cells, and which is attached to a marker, such as, for example, a fluorescent compound, or, for example, horseradish peroxidase or alkaline phosphatase. After an additional washing step, it is determined whether the secondary antibody has bound, for example by measuring light emission or by adding horseradish peroxidase or alkaline phosphatase substrate. Such detection methods are well known in the art.

In some embodiments, a CD43 peptide of the present invention or a compound that contains a CD43 peptide of the present invention is contacted with a fraction of the sample that is enriched in antibodies and/or immune cells. In some embodiments, said fraction is an in vitro B cell culture or an in vitro T cell culture. In some embodiments, a CD43 peptide of the present invention or a compound that contains a CD43 peptide of the present invention is contacted with antibodies and/or immune cells that are substantially purified from a biological sample, such as a purified B- cells, which is obtained by selecting CD19 positive cells and/or an antibody/B cell fraction, which is purified using an anti-Ig antibody or a protein A or G purification method. Protein A or G purification methods are well known in the art. techniques, and protocols and reagents are commercially available. In the context of the present description, the term immune cells that are substantially purified from the sample means that at least 80%, preferably at least 85%, more preferably at least 90% or at least 95% of the cells of the obtained fraction are immune. cells. The term antibodies that are substantially purified from the sample means that at least 80%, more preferably at least 85%, more preferably at least 90%, or at least 95%, by weight of the resulting fraction are antibodies.

Thus, the use of a CD43 peptide according to the present invention or the use of a compound that contains a CD43 peptide according to the present invention is also provided for binding and/or detecting an immune cell and/or an antibody or a functional portion or functional equivalent thereof. The specified immune cell and/or antibody or its functional part or functional equivalent in the preferred embodiment can specifically bind

- 15 040196 lymphoproliferative cells and/or myeloproliferative cells. Preferably, said myeloproliferative cells are AML cells or MDS cells or CML cells, preferably AML cells. The present document also provides a CD43 peptide according to the present invention or a compound that contains a CD43 peptide according to the present invention, for use as a fragment for the detection of AML-specific binding compounds such as antibodies and/or immune cells, as well as a method for determining the content in a sample of AML-specific antibodies and/or AML-specific immune cells, wherein said method comprises incubating a CD43 peptide according to the present invention or a compound that contains a CD43 peptide according to the present invention with said sample or with a fraction of said sample that contains antibodies and/or immune cells, and then determining binding by AML-specific antibodies and/or AML-specific immune cells of said CD43 peptide according to the present invention, or binding by AML-specific antibodies and/or AML-specific immune cells of said compound, which contains said CD43 peptide according to but a real connection. If such binding is found, then it is concluded that the specified sample contains AML-specific antibodies and/or AML-specific immune cells.

Also provided is a method for detecting the content of AML-specific antibodies and/or AML-specific immune cells in a sample, said method comprising incubating a CD43 peptide according to the present invention or a compound that contains a CD43 peptide according to the present invention with antibodies and/or immune cells that , essentially purified from the specified sample, and then determining the binding of AML-specific antibodies and/or AML-specific immune cells of the specified CD43 peptide according to the present invention, or the binding of AML-specific antibodies and/or AML-specific immune cells of the specified compound, which contains said CD43 peptide according to the present compound.

In some embodiments, the results of the detection studies described above are used to determine whether an individual has AML. If a sample obtained from an individual appears to contain AML-specific immune cells and/or AML-specific antibodies, then it can be concluded that said individual is an AML patient. Thus, the present document also provides a CD43 peptide of the present invention for use as a diagnostic agent, as well as a compound that contains a CD43 peptide of the present invention for use as a diagnostic agent. In addition, the use of the CD43 peptide according to the present invention for the diagnosis of AML is proposed, as well as the use of a compound that contains the CD43 peptide according to the present invention for the diagnosis of AML. Also provided is a diagnostic kit containing a CD43 peptide according to the present invention or a compound that contains a CD43 peptide according to the present invention, and means for detecting an antibody-bound CD43 peptide or an immune cell-bound CD43 peptide.

Such agents, for example, include labeled antibodies that are specifically directed against human antibodies or human immune cells. In some embodiments, said labeled antibodies are conjugated with horseradish peroxidase or alkaline phosphatase.

In some embodiments, a diagnostic kit is provided comprising a CD43 peptide of the present invention, or a compound that comprises a CD43 peptide of the present invention, and means for detecting an antibody or immune cell.

Such agents, for example, include labeled antibodies that are specifically directed against human antibodies or human immune cells. In some embodiments, said labeled antibodies are conjugated with horseradish peroxidase or alkaline phosphatase.

In some embodiments, a method is provided for typing a sample containing an antibody or a sample containing an immune cell, said method comprising contacting a CD43 peptide of the present invention (optionally in the context of an MHC (major histocompatibility complex) for T-cell detection) or a compound, which contains the specified CD43 peptide according to the present invention, with antibodies and/or immune cells of the specified sample and determining the binding of at least one of the specified antibodies and/or immune cells of the specified CD43 peptide sample according to the specified invention or the specified compound according to the present invention. If said CD43 peptide or said compound of the present invention is bound by antibodies and/or immune cells of said sample, said sample is typed as containing CD43-specific antibodies and/or immune cells.

In some embodiments, a method is provided for determining whether an individual has a myeloproliferative or lymphoproliferative disorder, preferably AML, said method comprising contacting a CD43 peptide of the present invention (optionally in the context of MHC for T-cell detection) or a compound that contains said

- 16 040196 CD43 peptide according to the present invention, with antibodies and/or immune cells of the specified individual and determining the binding of at least one of the specified antibodies and/or immune cells of the specified individual of the specified CD43 peptide according to the present invention or the specified compound according to the present invention. If said CD43 peptide or said compound of the present invention is bound by the antibodies and/or immune cells of said individual, then it is concluded that said individual has a lymphoproliferative or myeloproliferative disorder such as AML. In some embodiments, said CD43 peptide of the present invention, or said compound comprising said CD43 peptide of the present invention, is contacted with a sample that contains antibodies and/or immune cells from said individual, such as, for example, a blood sample or a bone marrow sample, or biopsy, such as, for example, myeloid sarcoma. In other embodiments, said CD43 peptide or compound of the present invention is contacted with a fraction of a sample obtained from said individual, said fraction containing immune cells and/or antibodies. In some embodiments, said CD43 peptide or compound of the present invention is contacted with antibodies and/or immune cells that are substantially purified from said sample, such as a purified fraction of B cells that is obtained by selecting CD19- positive cells and/or an antibody/B cell fraction that is purified using an anti-Ig antibody or protein A or G purification method.

In some embodiments, the results of the detection assays of the present invention are used to determine if an individual exhibits a detectable immune response against a myeloproliferative or lymphoproliferative disorder, such as AML. This is, for example, advantageous for determining whether a patient suffering from a myeloproliferative disorder who has received medical treatment, such as an AML patient who has received anti-AML treatment, such as an AML patient who has received immunotherapy, such as, for example, stem cell transplantation or infusion of donor lymphocytes, TPL reactions. To date, there are no diagnostic tools to test for the presence of a severe TPL reaction in a treated patient. Such a diagnostic tool is very necessary because, for example, 1) it will allow early identification of allogeneic SSC recipients with a high risk of relapse at a point in time before the onset of relapse, thus allowing procedures to be carried out at earlier stages, such as, for example, gradual reduction in the dose of immunosuppressants or infusion of donor lymphocytes; 2) it will allow dosed infusion of such donor leukocytes until antibodies against leukemia appear; and 3) it will provide reassurance to recipients of allogeneic TSC at times when they are often suffering from one of the many complications associated with TSC, where a severe TPL reaction may be present. Currently, patients have to wait and see if they relapse or not, and there is no way to predict the recurrence of the disease. Thus, the availability of a study to determine whether a patient has an immune response against AML will significantly improve the clinical care of patients undergoing TSC, influencing the prognosis of the disease and quality of life.

Thus, in some embodiments, a method is provided for determining whether an individual is eliciting an immune response against a myeloproliferative or lymphoproliferative disorder, preferably AML, said method comprising contacting a CD43 peptide of the present invention, optionally in the context of MHC, or a compound that contains said CD43 peptide according to the present invention with antibodies and/or immune cells of said individual and determining whether at least one of said antibodies and/or immune cells of said individual binds said CD43 peptide according to the present invention or said compound containing said peptide CD43 according to the present invention. If said CD43 peptide or said compound appears to be linked, this indicates that said individual is eliciting an immune response against said myeloproliferative or lymphoproliferative disorder, preferably AML. In some embodiments, it is determined whether the antibodies or B cells of the specified individual compete with the AT14-013 antibody for binding to CD43. Competing antibodies will be particularly effective against a myeloproliferative or lymphoproliferative disorder, preferably AML.

In some embodiments, an antibody, or a functional portion or functional equivalent thereof, that competes with an AT14-013 antibody for binding to CD43 is used to detect myeloproliferative cells in a sample. Thus, the use of an isolated recombinant or purified antibody, or a functional portion or functional equivalent thereof, that competes with the AT14-013 antibody for binding to CD43 is also provided to determine whether a sample contains myeloproliferative cells, preferably AML or MDS or CML- cells. Also provided herein is an isolated recombinant or purified

- 17 040196 an antibody or a functional part or a functional equivalent thereof that competes with the AT14-013 antibody for binding to CD43, for use in the diagnosis of a lymphoproliferative or myeloproliferative disorder, preferably AML or MDS or CML, as well as the use of an isolated recombinant or purified antibody or a functional portion or functional equivalent thereof that competes with the AT14-013 antibody for binding to CD43, for the diagnosis of AML. Also provided is the use of an isolated recombinant or purified antibody or a functional portion or functional equivalent thereof that competes with the AT14-013 antibody for binding to CD43 to produce a diagnostic agent for lymphoproliferative or myeloproliferative cells, preferably AML or MDS or CML cells. In some embodiments, a diagnostic kit is provided that contains an isolated recombinant or purified antibody, or a functional portion or functional equivalent thereof, that competes with the AT14-013 antibody for binding to CD43, and means for detecting an antibody-cell complex. These funds, for example, contain another antibody against AML cells, such as, for example, AT14-013. In some embodiments, these agents include labeled antibodies against another component of the cell surface of myeloid cells. In some embodiments, said agents include labeled antibodies against said antibody, or a functional portion or functional equivalent thereof, that competes with the AT14-013 antibody.

Also provided is an isolated recombinant or purified antibody or a functional portion or functional equivalent thereof that competes with the AT14-013 antibody for binding to CD43 for use as a diagnostic agent. In some embodiments, a method for determining the presence of myeloproliferative cells in a sample is provided, comprising contacting said sample with an isolated recombinant or purified antibody, or a functional portion or functional equivalent thereof, that competes with an AT14-013 antibody for binding to CD43, causing said antibody to bind, or its functional part or functional equivalent with myeloproliferative cells, if present, and determining the binding of myeloproliferative cells to the specified antibody or its functional part or functional equivalent, thereby determining whether myeloproliferative cells are present in the specified sample.

In some embodiments, said myeloproliferative cells are AML or MDS or CML cells.

In addition, the use of the AT14-013 antibody, or a functional portion or functional equivalent thereof, is also provided to determine the content of AML or MDS or CML cells in a sample. Also provided herein is the AT14-013 antibody or functional portion or functional equivalent for use in the diagnosis of AML or MDS or CML, as well as the use of the AT14-013 antibody or functional portion or functional equivalent for the diagnosis of AML or MDS, or CML. Also provided is the use of the AT14-013 antibody, or a functional portion or functional equivalent thereof, to produce a diagnostic agent for AML or MDS or CML.

Other interesting uses for the novel CD43 peptides of the present invention and, accordingly, for the nucleic acid molecules encoding them, or their functional equivalents, are prophylactic or semi-prophylactic uses and immunotherapy. In the context of the present description, the term semi-prophylactic use means that the individual already has a disease, but further progression of said disease is at least temporarily delayed or prevented. For example, a CD43 peptide according to the present invention or, respectively, a nucleic acid molecule encoding it or a functional equivalent thereof, or a vector containing a nucleic acid molecule or a functional equivalent according to the present invention, or a compound that contains a CD43 peptide according to the present invention, can be administered to an individual in a semi-prophylactic manner. suffering from moderate to high risk myelodysplastic syndrome (MDS). As described earlier herein, such a patient is at moderate to high risk of developing AML, so it is preferable for said patient to elicit an immune response against AML in advance using a CD43 peptide or a compound or nucleic acid molecule or a functional equivalent or a vector according to of the present invention before MDS progresses to AML. Another example of a semi-prophylactic use of a CD43 peptide of the present invention or a compound or nucleic acid molecule or functional equivalent or vector of the present invention is in patients with AML, MDS or CML who have undergone allogeneic hematopoietic stem cell transplantation (HSCT). The goal of such an allogeneic HSCT is to induce an allogeneic graft response against leukemia/MDS, but there is currently no approach to establish with certainty the development of such a reaction. The present invention provides the use of a CD43 peptide according to the present invention or, respectively, a nucleic acid molecule encoding it or a functional

- 18 040196 equivalent, or the use of a vector containing a nucleic acid molecule or a functional equivalent according to the present invention, or the use of a compound that contains a CD43 peptide according to the present invention, as a prophylactic or semi-prophylactic agent that induces an alloreactive immune response against CD43-expressing malignant cells (graft versus tumor response, eg against MDS, AML or CML). Another example of a prophylactic or semi-prophylactic use of a CD43 peptide of the present invention or a compound or nucleic acid molecule or a functional equivalent or a vector of the present invention is its use in patients with CML. Currently, CML is well controlled in many patients with tyrosine kinase inhibitors such as, for example, imatinib. However, the patient may develop resistance to at least one tyrosine kinase inhibitor. Moreover, the use of tyrosine kinase inhibitors is sometimes associated with adverse side effects such as edema, skin rash, fatigue, nausea, and myelosuppression. Tyrosine kinase inhibitors are also expensive. The present invention provides the use of a CD43 peptide according to the present invention or, respectively, a nucleic acid molecule or a functional equivalent encoding it, or the use of a vector containing a nucleic acid molecule or a functional equivalent according to the present invention, or the use of a compound that contains a CD43 peptide according to the present invention, in as a prophylactic agent or a semi-prophylactic agent that delays or prevents the progression of CML to AML. In some embodiments, said CD43 peptide, or compound, or nucleic acid molecule, or functional equivalent, or vector of the present invention is used in place of a tyrosine kinase inhibitor, for example, to reduce adverse side effects and/or reduce costs. In other embodiments, said CD43 peptide, or compound, or nucleic acid molecule, or functional equivalent, or vector of the present invention is co-administered with at least one tyrosine kinase inhibitor. Thus, the present document also provides a CD43 peptide according to the present invention or a compound that contains a CD43 peptide according to the present invention, or a nucleic acid molecule or a functional equivalent thereof encoding a CD43 peptide according to the present invention, or a vector containing a nucleic acid molecule or a functional equivalent according to the present invention, for use as a prophylactic agent or a semi-prophylactic agent. Also provided is the use of a CD43 peptide according to the present invention, or the use of a compound that contains a CD43 peptide according to the present invention, or the use of a nucleic acid molecule or its functional equivalent encoding a CD43 peptide according to the present invention, or the use of a vector according to the present invention, to obtain a prophylactic agent or semi-prophylactic an anti-AML agent, for example, for use in an AML patient who has undergone allogeneic HSCT. In some embodiments, said semi-prophylactic agent is for a patient with MDS or CML, as explained above. Said prophylactic agent or semi-prophylactic agent preferably contains a vaccine. In the context of the present description, the term prophylactic agent also includes semi-prophylactic agents.

In some embodiments, a CD43 peptide or compound or nucleic acid molecule or functional equivalent or vector of the present invention is used to treat a myeloproliferative or lymphoproliferative disorder, preferably AML. In the context of the present description, the term treatment includes the alleviation of at least one symptom and/or delay or even stop the progression of the disease, at least temporarily. In one preferred embodiment, a CD43 peptide according to the present invention, optionally in the context of MHC, or a nucleic acid molecule or, respectively, encoding a functional equivalent thereof, or a vector containing a nucleic acid molecule or a functional equivalent according to the present invention, or a compound that contains said CD43 peptide according to the present invention, is administered to an AML patient to stimulate his/her immune system, resulting in an improved immune response. In some embodiments, naïve T cells or B cells derived from an AML patient are cultured ex vivo and incubated with a CD43 peptide or a compound of the present invention, optionally in the context of MHC in the case of a T cell culture, to obtain AML-specific T β-cells or B-cells, which are then administered to the patient, optionally after they have been expanded ex vivo. In some embodiments, it is determined whether AML-specific B cells derived from a specified AML patient compete with the AT14-013 antibody for binding to CD43, since competing B cells would be particularly effective against AML, in particular because AT14-013 also targets leukemic stem cells, which are known to be more resistant to therapy and are often responsible for disease recurrence after treatment.

In some embodiments, adaptive cell therapy is used. In some embodiments, T cells obtained from an AML patient are tested for binding or activation

- 19 040196 using the CD43 peptide according to the present invention in the context of MHC or using a compound containing the CD43 peptide according to the present invention in the context of MHC, and T cells recognizing said CD43 peptide are preferably expanded ex vivo and further administered to the patient, leading to a T-cell response against AML.

In some embodiments, adaptive cell therapy with donor lymphocytes is used. Said donor T cells isolated from an AML patient who has undergone allogeneic HSCT or isolated from a HSCT donor are preferably tested for binding or activation using the CD43 peptide of the present invention in the context of MHC or using a compound containing the CD43 peptide according to the present invention in the context of MHC, and donor T cells recognizing said CD43 peptide are preferably expanded ex vivo and further administered to the patient, resulting in an allogeneic T cell response against AML.

In some embodiments, T cells are modified to provide them with an AML-specific binding moiety. Said T cells are preferably obtained from an AML patient or an MDS patient or a CML patient or a HSCT donor. In some embodiments, chimeric antigen receptor (CAR) T cells are obtained. These are T-cells with modified T-cell receptors that have a specific binding specificity of interest and are preferably derived from an antibody. Typically, CAR T cells are generated by fusion of single chain variable domains (scFv) derived from a monoclonal antibody and the transmembrane domain of CD3 zeta such that a zeta signal will be induced upon target scFv recognition.

In some embodiments, a CD43 peptide of the present invention, or, respectively, a nucleic acid molecule encoding it, or a functional equivalent, or a vector containing a nucleic acid molecule or a functional equivalent of the present invention, or a compound that contains a CD43 peptide of the present invention, is used to obtain and/or isolate a CD43-specific B cell and/or antibody, which, in turn, is used to obtain a modified T cell. For example, said CD43 peptide or compound or nucleic acid molecule or functional equivalent or vector is used to elicit, detect and/or isolate an AML-specific antibody or an AML-specific B cell. Then, in some embodiments, T cells are provided with the heavy chain and/or light chain variable domains of said antibody or B cell, thereby generating modified T cells with AML specificity. In some embodiments, such modified T cells are then administered to an AML patient, resulting in an AML-specific T cell response. In some embodiments, said modified T cells are CAR TAP cells. In some embodiments, said AML-specific antibodies or AML-specific B cells are tested for competition with the AT14-013 antibody for binding to CD43 before providing the T cells with the heavy chain and/or light chain variable domains of said antibodies or B -cells. Such competing antibodies are preferably chosen to generate modified T cells with AML specificity.

Thus, a CD43 peptide according to the present invention is also provided, optionally in the context of an MHC, or a compound which contains said CD43 peptide according to the present invention, or a nucleic acid molecule or a functional equivalent thereof encoding a CD43 peptide according to the present invention, or a vector containing the encoding molecule nucleic acid or functional equivalent according to the present invention, for use as a drug. Also provided is the use of a CD43 peptide of the present invention, optionally in the context of an MHC, or a compound that contains said CD43 peptide of the present invention, or a nucleic acid molecule or a functional equivalent thereof encoding a CD43 peptide of the present invention, or a vector containing a nucleic acid molecule or functional equivalent according to the present invention, to obtain AML-specific T-cells. In some embodiments, a method for producing a modified T cell is provided, the method comprising contacting an antibody-derived sample from an AML patient or a B-cell-containing sample obtained from an AML patient with a CD43 peptide or a compound of the present invention. which results in the production of anti-AML bound antibodies or B cells and then the production of at least one AML specific domain from an AML specific antibody or from an AML specific B cell obtained from said AML patient and providing a T cell at least one specified domain. In some embodiments, a method for producing a modified T cell is provided, the method comprising immunizing a non-human animal with a CD43 peptide, or a compound, or a nucleic acid molecule, or a functional equivalent, or a vector of the present invention, thereby eliciting an immune response against AML, and then obtaining at least one AML-specific domain from an AML-specific antibody, or

- 20 040196

An AML-specific B cell derived from said non-human animal, or obtaining at least one nucleic acid sequence encoding said at least one AML-specific domain, optionally after determining whether said AML-specific antibody competes or An AML-specific B cell with an AT14-013 antibody for binding to CD43, and providing the T cell with said at least one domain or said at least one nucleic acid sequence. Also provided herein is a CD43 peptide or compound of the present invention for use in immunotherapy, as well as a CD43 peptide of the present invention in the context of MHC for use in immunotherapy, as well as a nucleic acid molecule or a functional equivalent thereof that encodes a CD43 peptide of the present invention for applications in immunotherapy. In some embodiments, a vector containing a nucleic acid molecule or a functional equivalent of the invention is provided for use in immunotherapy. In addition, the use of a CD43 peptide according to the present invention, or the use of a compound that contains the CD43 peptide of the present invention, or the use of a nucleic acid molecule or its functional equivalent encoding a CD43 peptide of the present invention, or the use of a vector containing a nucleic acid molecule or a functional an equivalent according to the present invention for the preparation of a medicament for a myeloproliferative or lymphoproliferative disorder, preferably against AML.

Also provided is an immunogenic composition containing a CD43 peptide according to the present invention and/or containing a compound that contains a CD43 peptide according to the present invention and/or containing a nucleic acid molecule or its functional equivalent encoding a CD43 peptide according to the present invention and/or containing a vector which contains a nucleic acid molecule or a functional equivalent according to the present invention. Said immunogenic composition preferably further comprises a biocompatible additive such as, for example, a carrier, diluent, excipient or excipient. In some embodiments, a vaccine is provided comprising the CD43 peptide of the present invention, optionally in the context of MHC. In some embodiments, a vaccine is provided comprising a compound that comprises said CD43 peptide of the present invention, and a vaccine comprising a nucleic acid molecule or a functional equivalent thereof encoding a CD43 peptide of the present invention, and a vaccine comprising a vector that comprises a nucleic acid molecule or functional equivalent according to the present invention. In another embodiment, there is provided a composition comprising a CD43 peptide of the present invention, or a composition comprising a compound comprising a CD43 peptide of the present invention, or a composition comprising a nucleic acid molecule or a functional equivalent thereof encoding a CD43 peptide of the present invention, or a composition, containing a vector that contains a nucleic acid molecule or a functional equivalent according to the present invention, while the specified composition is a pharmaceutical composition, which also contains a pharmaceutically acceptable carrier, diluent or excipient.

In some embodiments, for the treatment of a myeloproliferative or lymphoproliferative disorder, in the preferred embodiment of AML, an isolated recombinant or purified antibody or a functional portion or functional equivalent thereof that competes with the AT14-013 antibody for binding to CD43 is used. As described in WO 2015/093949, the AT14013 antibody was obtained from an AML patient in complete remission, demonstrating that AT14-013 is effective against AML. In addition, AT14-013 also targets leukemia stem cells, which are known to be more resistant to therapy and are often responsible for disease recurrence after treatment. Thus, antibodies that compete with AT14-013 for binding to CD43 will also be very effective against myeloproliferative disorders like AML. Therefore, administration of such antibodies to an AML patient will effectively neutralize and/or kill AML cells. Thus, in some embodiments, an isolated recombinant or purified antibody, or a functional portion or functional equivalent thereof, that competes with the AT14-013 antibody for binding to CD43 is provided for use as a drug. In some embodiments, the use of an isolated recombinant or purified antibody, or a functional portion or functional equivalent thereof, that competes with an AT14-013 antibody for binding to CD43 is provided to produce a drug.

Also provided is an isolated recombinant or purified antibody, or a functional portion or functional equivalent thereof, that competes with the AT14-013 antibody for binding to CD43, for use in a method for at least partial treatment or prevention of a myelodysplastic or myeloproliferative or lymphoproliferative disorder, as well as the use of an isolated recombinant or purified antibody, or a functional portion or functional equivalent thereof, that competes with the AT14-013 antibody for binding to CD43 to produce a drug against myeloproliferative or lymphoproliferative races

- 21 040196 construction. Said myeloproliferative disorder preferably includes AML. In other embodiments, a composition is provided comprising an isolated recombinant or purified antibody, or a functional portion or functional equivalent thereof, that competes with the AT14-013 antibody for binding to CD43. The specified composition in the preferred embodiment is a pharmaceutical composition that contains a pharmaceutically acceptable carrier, diluent or excipient.

As previously described herein, in some embodiments, there is provided an isolated synthetic or recombinant nucleic acid molecule, or a functional equivalent thereof, encoding a CD43 peptide of the present invention. Such a nucleic acid molecule or functional equivalent is, for example, suitable for the production of the CD43 peptide of the present invention using a nucleic acid expression system such as, for example, host cells. In some embodiments, AML cells are used as host cells. In some embodiments, THP-1 cells are used as host cells. In some embodiments, host cells are selected from the group consisting of Kasumi 3 cells, HL60 cells, KG1a cells, SH2 cells, MonoMac6 cells, Molm13 cells, and K562 CML cells.

The specified nucleic acid molecule or functional equivalent according to the present invention is also suitable for obtaining an immune response. For example, a nucleic acid molecule or a functional equivalent according to the present invention is administered to an AML patient in order to induce or enhance an AML-specific immune response (immunotherapy). In some embodiments, a nucleic acid molecule or functional equivalent of the present invention is administered to a patient with MDS or CML in order to delay or prevent the progression of MDS or CML to AML (prophylactic or semi-prophylactic uses). In some embodiments, a nucleic acid molecule or functional equivalent of the present invention is administered to a non-human animal to elicit an anti-AML immune response, after which AML-specific antibodies and/or AML-specific immune cells can be harvested from said animal. In an alternative embodiment, nucleic acid sequences encoding variable regions of AML-specific B cells derived from said non-human animal are sequenced to obtain at least one AML-specific variable domain nucleic acid sequence, after which at least one nucleic acid molecule an acid containing sequences of an AML-specific variable domain is introduced into producing cells to form AML-specific antibodies.

In some embodiments, the nucleic acid molecule or functional equivalent of the present invention is contained in a gene delivery vehicle that facilitates the introduction of said nucleic acid molecule or functional equivalent into the cell of interest. Thus, a gene delivery vehicle is also provided, preferably a vector containing a nucleic acid molecule or a functional equivalent according to the present invention. Also provided herein is a host cell comprising a nucleic acid molecule or a functional equivalent according to the present invention and/or comprising a carrier or gene delivery vector according to the present invention.

Although the present application may describe the features as part of the same implementation or as part of separate implementations, the scope of the present invention also includes implementations containing any combination of all or some of the features described here.

The present invention is further explained in the following examples. These examples do not limit the scope of the present invention, but simply serve to explain the present invention.

Brief description of the drawings

Fig. 1 shows the sequence of AT14-013 (2K23-1K13) containing the heavy and light chain variable sequences and antibody CDR sequences.

Fig. 2 - Binding of AT14-013 to AML cell lines and freshly isolated primary AML blasts from newly diagnosed patients. FAB: Franco-American-British AML classification (Bennett et al., 1976).

Fig. 3 - AT14-013 binds to AML cell lines and primary isolated AML cells. Typical examples of binding of AT14-013 from patient 101 to the AML Kasumi3, SH-2, Molm13 and THP-1 cell lines and to primary leukemic blasts isolated from newly diagnosed AML patients (FAB classification M0-M5). A self-produced human influenza-specific antibody was used as a negative control (gray histograms).

Fig. 4-AT14-013 also binds to leukemia blasts from patients with high risk myelodysplastic syndrome (MDS/RAEB I and II) and chronic myeloid leukemia (CML) blast crisis. Typical examples are presented; 22 040196 patient identification codes are given, with the exception of K562, which is a CML cell line. Self-produced human influenza antibodies (gray histograms) were used as a negative control. *BL-060: Biphenotypic leukemia responding well to AML treatment.

Fig. 5 - AT14-013 does not bind to non-myeloid cells. (a) AT14-013 did not bind to healthy PBMCs, T cells (CD3+), B cells (CD19+), non-activated monocytes (CD14+), or primary isolated thymocytes (with the exception of a small population of myeloid cells that are present in the fetal thymus). ), (b) AT14-013 also did not bind primary isolated B or T-ALL cells, lymphoma or multiple myeloma cells. (c) AT14-013 also did not bind colon carcinoma cell lines or isolated cells derived from patients with colon carcinoma (Colon CA) or healthy colon or ileum. AT14-013 binds to granulocytes (a) and human melanoma cell lines (c). Self-produced human influenza antibodies (gray histograms) were used as a negative control.

Fig. 6 - CDC and ADCC. Calcein-labeled THP-1 cells were incubated with AT14-013 and rabbit serum complement. Living cells were identified as calcein+, dapi cells. Using the author's research method based on the study of chains of granules, the number of dead cells can subsequently be calculated as a measure of complement-dependent cell death (CDC). Incubation of THP1 cells with CD33 did not induce CDC (left panel). AT14-013 can also induce antibody mediated cellular cytotoxicity (ADCC) in the Jurkat reporter system using the AML SH-2 cell line or freshly isolated leukemic blasts as target cells (right panel).

Fig. 7 - AT14-013 target identification: immunoprecipitation (IP). IP using biotin labeled (via sortase tag) AT14-013 lysate from THP1 cells resulted in a ~140 kD band on Imperial Coomassie stained gel. The band is specific as it is not visible on IP using AT10-002 THP1 cell lysate or Jurkat cell lysate. The band was removed from the gel and the target was identified as CD43 by mass spectrometry.

Fig. 8 - confirmation of the target AT14-013. Lysates of THP-1 and Molm13 cells were immunoprecipitated using AT14-013 or influenza specific antibody AT10-002. Immunoblotting using a mouse anti-CD43 antibody (clone Mem59) confirmed that CD43 is the target for AT14-013 binding.

Fig. 9 - AT14-013 binds to a unique CD43 epitope. (a) THP-1 cells were stained with commercially available CD43-specific antibodies DF-T1, 84-3C1, L10 and Mem59 and with AT14-013. All antibodies bound to the membrane of THP-1 cells. (b) AT14-013 has a different binding profile compared to commercially available CD43-specific antibodies. Kim ea (Kim et al., 2014) summarizes the binding of commercially available anti-CD43 YG5, 2C8, 8E10, and DFT-1 antibodies to various cell lines. The authors of the present invention compared the binding of AT 14-013 with the same cell lines and found a different binding profile. (c) The competition experiment was performed using AT14-013 and commercially available CD43-specific antibodies. Briefly, THP-1 cells were incubated with the indicated antibodies at increasing concentrations, after which a possibly competing antibody (referred to as a competing antibody) was added. AT14-013 binding to THP-1 target cells was not affected by pre-incubation of the cells with commercially available anti-CD43 antibodies, whereas such commercially available anti-CD43 antibodies inhibited each other's binding to THP-1 cells. The results are shown for experiments in which AT14-013 or 84-3C1 is a competitive antibody, (d) Brief description of competition experiments. AT14-013 does not compete with commercially available anti-CD43 antibodies for binding to THP-1, indicating that AT14013 binds a different epitope.

Fig. 10 - deglycosylation of THP-1 cells using neuraminidase (sialidase) removes sialic acids from the cell membrane. No indicates no neuraminidase treatment and 1:20 and 1:200 indicates a dilution of neuraminidase. Antibodies AT14-013, Mem59, DF-T1 and 84-3C1 lost their ability to bind to THP-1 cells after treatment of these cells with neuraminidase. Clone L10 does not bind to the sialylated CD43 epitope because treatment of THP-1 cells with neuraminidase does not affect the binding of this antibody to its target cells.

Fig. 11 - CD43 truncated variants map the epitopes of commercially available DF-T1 and MEM59 antibodies. a) Immunoblot of HEK293T cells expressing truncated CD43 variants probed with an anti-CD43 antibody directed to the intracellular C-terminal tail (segment) of said protein. b) Immunostaining of the same blot using CD43-specific antibodies MEM59 (upper panel) and DF-T1 (lower panel) revealed the presence of their epitope in region C (amino acids 59-82).

Fig. 12 - Immunoprecipitation of truncated CD43 variants from THP1 cells identifies the AT14-013 epitope. a) Immunoblot of entry lysates of sorted CD43 truncated THP1 cells probed with anti-Flag antibody. Ponseau S coloring

- 23 040196 shows equal sample loading. All mutant proteins are expressed. b) Anti-Flag immunoblot of eluted immunoprecipitates of AT14-013 THP1 cell line variants shows binding to A-F mutants and no binding to epitope-determining H-J mutants. c) Immunoblot using anti-CD43 antibody binding its cytoplasmic tail (Novus) showing endogenous immunoprecipitated CD43 in all samples, as well as staining of truncated variants.

Fig. 13 - amino acid sequence of CD43 (Genbank CCDS10650.1). The signal peptide, AT14-013 epitope, transmembrane domain, and intra- and extracellular domains are indicated.

Fig. 14 - binding of AT14-013 to other AML blasts. Binding of AT14-013 to freshly isolated primary AML blasts (CD45dim) from newly diagnosed patients. A self-produced human influenza-specific antibody was used as a negative control. For commercial mouse anti-CD43 antibodies, a mouse anti-cytomegalovirus (CMV) antibody was used as a control.

WHO: Swerdlow S.H. WHO classification of tumors of hematopoietic and lymphoid tissues (2008). CD43+ T cells and tonsil cells were used as an additional control for the assay. AT14-013 does not bind these healthy cells.

(interval in % = -; <10%, + 10-25%, ++ 25-50%, +++ 50-75%, ++++ 75-100%).

Fig. 15 - ADCC and CDC. a) AT14-013 (open squares) is able to induce antibody-mediated cellular cytotoxicity (ADCC) in the AML SH-2 cell line with PBMC cells at an effector to target ratio of 50:1. Living cells were identified as calcein+, dapi cells. Using the author's method of research, based on the study of chains of granules, it is subsequently possible to calculate the number of dead cells. Incubation of SH2 cells with AT10-002 did not induce ADCC (black dots). The calculated EC ₅₀ concentration for AT14-013 was 0.16 μg/ml. Calcein-labeled SH-2 cells (b) were incubated with AT14-013 (open squares) or AT10-002 (black dots) and rabbit serum complement. Living cells were identified as calcein+, dapi cells. Incubation of SH2 cells or AML blasts with AT10-002 did not induce CDC (black dots). The calculated EC ₅₀ concentration for AT14-013 was 1.86 μg/ml.

Fig. 16-a) Anti-Flag immunoblot of eluted immunoprecipitates of AT14-013 THP1 cell line variants shows binding to A-F2 mutants, less binding to G mutant, and no binding to H-J mutants. b) Immunoblot using anti-CD43 antibody binding its cytoplasmic tail (Novus) showing endogenous immunoprecipitated CD43 in all samples, as well as staining of truncated variants. This control confirms that immunoprecipitation was successful for all submitted samples.

Fig. 17-a) Treatment of mice inoculated with SH-2 AML cells resulted in a 90.3% inhibition of tumor growth as determined by whole body measurements of sacrificed mice (p<0.001, repeated analysis of variance (ANOVA)). b) The number of AML cells, measured by the number of photons per minute (cpm), shows a strong decrease in all measured organs (p=0.0011, ANOVA in duplicate). c) Estimation of the number of tumor cells by fluorescence-activated cell sorting (FACS) in bone marrow and liver (p=0.0017, duplicate ANOVA).

Fig. 18 - typical examples of AT14-013 binding to fetal CD34+ hematopoietic stem cells (HSCs), but not to fetal CD34+CD38+ progenitors or fetal CD34-CD38 mature cells. Gray bar graphs: Influenza control antibody AT10-002 described in WO 2013/081463.

Fig. 19 - AT14-013 reacts with autologous leukemic stem cells. Donor 101 AML blasts (the same donor from which AT14-013 producing B cells were obtained) were stained with AT14-013 and antibodies specific for CD34 and CD38, and with antibodies against CD45 (BD, catalog number 348815 ) to distinguish the total blast population (CD45 dim) from healthy cells in the bone marrow and analyzed by flow cytometry.

Examples

Example 1

Materials and methods.

Materials obtained from patients and healthy people.

The study protocols were approved by the Medical Ethics Committee of the Academic Medical Center. All participants signed an informed consent. Participants included healthy individuals and patients with hematological malignancies from the clinic to which the authors of the present invention are related, who were donors of peripheral blood and/or bone marrow.

Obtaining AML-specific clone AT14-013.

As described in example 2 of WO 2015/093949, transduced naïve and IgG-synthesizing AML patient 101 memory B cells immortalized by administration of Bcl6 and Bcl-xL described previously (Kwakkenbos et al., Nat. Med., 2010 and Example 1 publication WO 2015/093949), were sown

- 24 040196 at a concentration of 20 or 40 cells per well (hereinafter referred to as microcultures) and propagated using IL-21 and CD40L. The supernatants of the propagated B-cell microcultures were then screened for antibody binding to AML cell lines (among others THP 1, MonoMac) and liver and colon cell lines by FACS using human IgG H+L AF647 (Life Technologies) or human IgG- PE (Southern Biotech) as a secondary antibody. Some self-produced antibodies such as anti-CD30 (expressed on the surface of activated B- and T-lymphocytes), anti-CD33 (expressed on the surface of monocytes, myeloid progenitor cells and leukemia myeloid cells) were used as negative controls. cells), the D25 antibody (against the RSV virus (RSV) described in WO 2008/147196) and the AT10-002 antibody (against influenza as described in WO 2013/081463). Microcultures that bind to AML cell lines but not to liver and colon cell lines were selected and plated at 1 cell/well, and their supernatants were re-examined for specificity to AML cell lines. Clones with supernatants were chosen for sequencing that specifically bound AML cell lines but did not bind liver or colon cell lines, or healthy PBMC cells and bone marrow cells. Clones were propagated under normal culture conditions in the presence of fetal bovine serum (FBS) depleted of IgG (Hyclone) and antibodies purified from these culture supernatants as described below for recombinant antibodies. The recombinant antibodies are then re-examined for specific binding. One of the obtained AML-specific antibodies was the antibody AT14-013. The detected AT14-013 antibody was further tested on many freshly isolated blasts from newly diagnosed AML patients (FAB M0-M5) to determine binding using human IgG H+L AF647 (Life Technologies) as a secondary antibody.

Cloning of the AML-specific antibody AT14-013.

As described in Example 1 of WO 2015/093949, in order to produce a recombinant antibody, the present inventors isolated total RNA using the RNeasy® mini kit (Qiagen), prepared cDNA, performed PCR, and cloned the heavy and light chain variable regions into the pCR2 cloning vector. 1TA (Invitrogen). In order to exclude mutations induced by reverse transcriptase or DNA polymerase, the inventors carried out several independent cloning experiments. To obtain a recombinant mAb, the heavy and light chain variable regions of each antibody were cloned in-frame with human IgG1 and Kappa constant regions into a pcDNA3.1-based vector (Invitrogen) and transiently transfected 293T cells. Recombinant antibodies from the culture supernatant were purified using protein A or G, depending on the Ig subtype of the clone.

CDC and ADCC.

In order to quantify the complement-dependent cell death (CDC) of target cells induced by the AML-specific antibody AT14-013, the inventors used a FACS-based leukemic cell lysis assay. THP-1 cells were incubated with 2 μM Calcein AM (Becton Dickinson) for 30 min at 37°C. Calcein-labeled THP-1 cells were incubated with antibodies and rabbit serum complement for 4 h at 37°C. FACS calibration microcarriers (Accudrop Fluorescent Beads, BD Biosciences) were added to the indicated cells in a 50/50 ratio, after which a standard amount of microcarrier was obtained using the FACS method. Since the equal volume taken for analysis was established using calibration microcarriers, the number of dead cells was calculated as 100 - ((Dapi-negative, Calcein AM positive cells with appropriate treatment / Dapi-negative, Calcein AM positive cells in control) x 100). For antibody-mediated cellular cytotoxicity (ADCC), a readout system was prepared using Jurkat cells that stably transduced NFAT(6x)-IL2 (minimal promoter)-GFP and CD16A (FcR-IIIa). Activation of the CD16A receptor by a bound antibody in this system activates the nuclear factor of activated T cells (NFAT), which induces the expression of green fluorescent protein (GFP), which is then used as a reader to quantify the activation of effector cells. AML cells (target cells) were incubated with antibodies and mixed with Jurkat cells (effector cells) which were stained with Calcein AM as described above. The effector:target ratio was 1:1.

Identification and validation of the AT14-013 target.

THP-1 cells were lysed (mixture of 0.5% Triton X114 (Sigma), 150 mM NaCl, 10 mM Tris-HCl pH 7.4, 1.5 mM MgCl ₂ supplemented with protease and phosphatase inhibitors (Roche)) and prepurified with an irrelevant antibody (self-produced anti-RVS antibody D25), protein-G beads, and streptavidin (Pierce) to remove non-specifically binding proteins. The prepurified lysate was then incubated with AML-specific bead-bound antibodies or influenza-specific antibody AT10-002 as a negative control (3 h at 4°C). The antibody-incubated beads were washed five times in lysis buffer,

- 25 040196 supplemented with 0.5% deoxycholate and 0.1% sodium dodecyl sulfate (SDS), bound proteins were eluted from the beads (0.1 M glycine pH 10.5, 150 mM NaCl, 1% Triton X100, 1 mM EDTA), and then electrophoresis was performed in polyacrylamide gel (PAAG) in the presence of sodium dodecyl sulfate (SDS). 85% of the resulting IP samples were subjected to SDS-PAGE and stained with Imperial Protein Strain (Pierce) to stain total protein and remove special bands for mass spectrometry. The remaining IP samples were subjected to SDS-PAGE and transferred to a PVDF membrane (Bio-RAD) for immunoblotting. The blot was stained with Ponseau S for total protein detection and blocked with bovine serum albumin (BSA), then incubated with mouse anti-CD43 antibody (clone MEM-59, Abcam) for Western blot analysis.

Epitope mapping: competition.

THP-1 cells were pre-incubated for 60 min on ice with AT14-013 and commercially available anti-CD43 antibodies: PE mouse anti-human CD43 antibody (Ebioscience, clone 84-3C1), FITC mouse anti-human CD43 antibody (Invitrogen, clone L10) FITC mouse anti-human CD43 (Abcam; clone MEM-59), mouse unlabeled anti-human CD43 (Abcam; clone MEM-59), mouse unlabeled anti-human CD43 (Thermo Scientific; clone DF-T1). The maximum concentration of blocking antibodies was 10 μg/ml. Competitor antibody was then added at a final concentration of 1 μg/ml. At this stage, the final concentration of these blocking antibodies is 2 μg/ml. The cells were incubated for 30 min on ice, after which the dapi dye (Sigma) was added to exclude the analysis of dead cells. Samples were analyzed by flow cytometry.

Epitope mapping: deglycosylation.

THP-1 cells were incubated with neuraminidase (Roche, dilution 1:20 or 1:200) for 60 min at 37°C to remove sialic acids from the surface of CD43 (de Laurentiis et al., 2011). Cells were then washed, blocked in 60% normal goat serum and incubated with AT14-013 and commercially available anti-CD43 antibodies DF-T1, 84-3C1, L10 and MEM-59 as described above. In order to compare the staining of cells with different fluorochromes, binding to untreated cells (without neuraminidase) was taken as a unit. In FIG. 10 shows the fold increase/decrease in binding to neuraminidase-treated cells.

Epitope mapping: truncated variants of CD43.

CD43 cDNA was obtained from Geneart (Life Technologies) and adapted to contain the 3xFLAG tag in frame in either the C-terminal region or the N-terminal region (the C-terminal region of the signal peptide containing the first 19 amino acids of CD43) . The cDNA was cloned into a third generation lentiviral vector pHEF-TIG containing IRES-GFP 3' CD43 cDNA; VSV-G lentiviral particles were produced in HEK293T cells. THP1, MOLM and other cells were transduced with these viruses in the presence of retronectin and sorted using GFP to obtain a pure population of cells overexpressing CD43. Truncated CD43 variants were constructed by NDP cloning of CD43 cDNA with a C-terminal FLAG tag to produce a signal peptide (AA 1-19) followed by the full-length wild-type extracellular sequence (variant A: S20-P400 followed by 3xFLAG: DYKDHDGDYKDHDIDYKDDDDK ) or truncated extracellular sequences (BJ variant). B: 31-400; C: 59-400; D: 82-400; E: 112-400; F: 133400; G: 166-400; H: 184-400; I: 202-400; J: 220-400 (transmembrane domain starts at AA 255). These variants were expressed in THP1 cells by lentivirus transduction and GFP sorting. Sorted cells were lysed and immunoprecipitated using AT14-013 and controls as described above. Eluted IP samples were subjected to SDS-PAGE and immunoblotting using an anti-FLAG-HRP antibody (Sigma) to detect binding.

Results.

AT14-013 binds specifically to AML cells.

In this example, the target is identified for the AML-specific antibody AT14-013, which was recently developed in the laboratory to which the authors of the present invention are related (publication WO 2015/093949 and Fig. 1). This antibody is from a patient referred to as patient 101. He was diagnosed with intermediate-risk AML (no cytogenetic or molecular abnormalities, FAB classification AML-M5) at 49 years of age. He received two courses of chemotherapy (cytarabine, idarubicin, amsacrine) and one course of anchoring chemotherapy (busulfan, cyclophosphamide) followed by autologous hematopoietic stem cell transplantation (HSCT) because there was no available MHC-compatible related stem cell donor. Fourteen months after the first diagnosis, he suffered a relapse of the disease. He achieved complete remission after one cycle of high-dose cytarabine, after which he underwent reduced-intensity allogeneic HSCT from an unrelated MHC-matched donor (RIST-MUD). Six weeks later, he developed acute GVHD of the skin, liver, and intestines (stage 1, class II), which responded well to corticosteroid therapy. Considering the fact that this patient remained healthy for more than 5 years, despite the high risk of his disease,

- 26 040196 it can be considered that this patient developed a graft-type reaction against AML, which formed the basis for choosing a patient to search for an effective AML-specific humoral immune response. B-cells were isolated from a phlebotomy product obtained from this patient 38 months after HSCT, immortalized by the introduction of Bcl6 and Bcl-xL, as described previously (Kwakkenbos et al., Nat. Med., 2010), and cultured at a concentration of 20 or 40 cells/well. The supernatants of these microcultures were screened for binding to AML cell lines and AML specific microcultures subcloned at a single cell/well concentration. One of the antibodies identified by this procedure is AT14-013, an IgG-κ, highly somatic hypermutated antibody.

AT14-013 specifically binds to a wide range of AML cell lines and primary AML cells, covering all FAB classifications of AML as shown in FIG. 2. In FIG. 3 shows several representative examples of AT14-013 binding to Kasumi3, SH-2, Molm13 and THP-1 cells and primary leukemic blasts isolated from newly diagnosed AML patients. In addition, AT14-013 binds to other myeloid malignancies such as AML derived from patients with high-risk myelodysplastic syndrome (MDS/RAEB VII) or chronic myeloid leukemia (CML) blast crisis and the CML K562 cell line (Fig. 4). AT14-013 showed some binding to granulocytes, but did not bind to normal peripheral blood mononuclear cells (PBMC), bone marrow cells, thymocytes, hematological lymphatic lineage malignancies, or healthy or malignant liver and colon cells. AT14-013 associated with cultured melanocytes and melanoma cell lines (Fig. 5).

AT14-013 induces CDC and ADCC in target cells.

AT14-013 can induce complement-dependent cytotoxicity and antibody-mediated cellular cytotoxicity (FIG. 6) of AML cell lines and primary isolated AMLblasts.

The target of T14-013 is the unique CD43 epitope.

Next, the authors of the present invention determined the target AT14-013. Immunoprecipitation (IP) of a THP-1 lysate incubated with AT14-013 labeled biotin containing sortase as a label resulted in a ~140 kDa band. The band is specific as it is not visible on IP using AT10-002 THP1 cell lysate or Jurkat cell lysate (FIG. 7). Mass spectrometric analysis of the immunoprecipitate band detected CD43 as the target protein. Three of the three expected intracellular peptides were identified, providing 7% protein coverage, extracellular peptides were not identified because they are highly glycosylated. Binding of CD43 by AT14-013 was confirmed by Western blot analysis. Briefly, THP-1 and Molm13 cell lysates were immunoprecipitated using AT14-013 or influenza specific antibody AT10-002. Western blot analysis using mouse anti-CD43 antibody (clone Mem59) confirmed that CD43 is the target for AT14-013 binding (FIG. 8).

CD43 is highly expressed on the surface of healthy and malignant cells. CD43-specific antibodies can be made and are also commercially available, such as DF-T1, 84-3C1, L10 and MEM-59. With these antibodies, the expression of CD43 by THP-1 cells was confirmed (FIG. 9A). The observation that AT14-013 does not bind to non-myeloid cells and the different binding profile of AT14-013 to all cell types and cell lines compared to other anti-CD43 antibodies (Fig. 9B) suggests that AT14-013 recognizes a different CD43 epitope than other anti-CD43 antibodies. Indeed, when competition experiments were performed with incubation of THP-1 cells with commercially available anti-CD43 antibodies and AT14-013 antibody, the present inventors found that these anti-CD43 antibodies compete with each other for binding to THP-1, but not to AT14-013 (FIGS. 9C and 9D). It should be noted that CD43 clones L10 and 84-3C1 have been described as competing with each other (L. Borche et al., 2005); the same was confirmed in the experiment of the inventors.

The CD43 protein is a highly glycosylated protein (de Laurentiis et al., 2011). Anti-CD43 antibodies Mem59, DF-T1 and 84-3C1 (but not L10) bind to the sialylated epitope because after pretreatment of target cells with neuraminidase, which removes all α-Nacetylneuraminic acids (sialic acids), these antibodies lose their binding ability with CD43 (US Patent No. 2010/0234562 A1). In FIG. 10 shows that the ability of AT14-013 to bind to THP-1 cells is also lost upon pre-incubation of THP-1 cells with neuraminidase, indicating that AT14-013 specifically binds to the sialylated CD43 epitope.

To more accurately identify the AT14-013 binding epitope, the present inventors generated 10 extracellular truncated Flag-tagged CD43 variants that were expressed in HEK and THP1 cells. Western blot analysis of lysates of these cells, incubated with Mem59 or DF-T1, confirmed the binding of these antibodies to a similar epitope located between amino acids 59-82 (Fig. 11A, B). Investigated the binding of AT14-013 using cell immunoprecipitation

- 27 040196

THP1 transduced by these truncated variants. AT14-013 interacts strongly with variants A-F, to a lesser extent with variant G, and does not interact with variants H-J, as shown in the IP immunoblot using an anti-Flag antibody (Fig. 12A, B). In FIG. 12C was confirmed by IP AT14-013 using an antibody against the C-terminal region of CD43. All samples contained endogenous CD43 while they contained only truncated CD43 present up to variant G. Thus, the inventors concluded that the AT14-013 epitope was between amino acids 133 and 184.

Example 2 Binding to AML blasts.

Materials and methods.

Binding of the AT14-013 antibody to various cells was examined using the procedure described in Example 1 under the heading Obtaining an AML-Specific Clone AT14-013. Patient samples were stained with anti-human CD45 antibody (BD) prior to analysis. AML cells were defined as CD45dim. Healthy PBMC cells were stained with an anti-human CD3 antibody (biolegend). Tonsil-derived polymorphic nucleated cells were isolated using a ficoll density gradient.

Results.

AT14-013 specifically binds to a wide range of AML cell lines and primary AML cells, covering all FAB AMLs as shown in Example 1 and FIG. 4. In addition, the antibody was tested on a wider panel of AML blasts. It has been shown to bind to all AML blasts investigated to date, and often better than commercial anti-CD43 antibodies. Interestingly, the sialic acid-independent L10 antibody bound in at least almost all samples. In addition, antibodies were tested in healthy, CD43-expressing T cells and tonsil-derived cells. In this case, staining was observed only in commercial antibodies. The summarized results are shown in Fig. 14.

Example 3 ADCC and CDC.

In addition to Example 1 and FIG. 6, another experiment was performed to determine ADCC and CDC.

Materials and methods.

To quantify antibody-mediated cellular cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC) of target cells induced by AML-specific antibody AT14-013, the inventors used a FACS-based leukemic cell lysis assay. SH2 cells were incubated with 10 nM Calcein AM (Becton Dickinson) for 30 min at 37°C. The calcein-labeled cells were then incubated with antibodies and healthy peripheral blood mononuclear cells (PBMC, effector:target ratio 50:1) for 4 h or rabbit serum complement for 1 h at 37°C. Calibration microcarriers for FACS (Accudrop Fluorescent Beads, BD Biosciences) were added to the indicated cells in a ratio of 50/50, after which a standard amount of microcarrier was obtained using the FACS method. Since an equal volume taken for analysis was established using calibration microcarriers, the number of dead cells was calculated as: 100).

Results.

AT14-013 induces CDC and ADCC in target cells.

AT14-013 can induce antibody-mediated cellular cytotoxicity (FIG. 15A) and induce complement-dependent cytotoxicity (FIG. 15B) of AML cell lines and primary isolated AML blasts.

Example 4 Epitope Mapping: Truncated Variants of CD43.

In addition to Example 1 and FIG. 12 further explored the AT14-013 binding epitope.

Materials and methods.

Methods similar to Example 1 were used. CD43 cDNA was obtained from Geneart (Life Technologies) and adapted to contain the 3xFLAG tag in frame either at the C-terminal region or at the N-terminal region (C-terminal region of the signal peptide containing the first 19 amino acids of CD43). The cDNA was cloned into the third generation lentiviral vector pHEFTIG containing IRES-GFP 3' CD43 cDNA; VSV-G lentiviral particles were produced in HEK293T cells. THP1, MOLM and other cells were transduced with these viruses in the presence of retronectin and sorted using GFP to obtain a pure population of CD43-transduced cells. Truncated CD43 variants were constructed by PCR cloning to CD43 DNA with a C-terminal FLAG tag to generate a signal peptide (AA 1-19) followed by the full-length wild-type extracellular sequence (variant A: S20-P400 followed by 3xFLAG: DYKDHDGDYKDHDIDYKDDDDK) or truncated extracellular sequences (BJ variant). B: 31-400; C: 59-400; D: 82-400; E: 112-400; F: 133-400; F2: 148-400; G: 166-400; H: 184-400; I: 202-400; J: 220-400 (transmembrane domain starts at AA 255). These variants were expressed in THP1 cells

- 28 040196 using lentivirus transduction and sorting using GFP. Sorted cells were lysed and immunoprecipitated using AT14-013 and controls as described above. Eluted IP samples were subjected to SDS-PAGE and immunoblotting using an anti-FLAG-HRP antibody (Sigma) to detect binding.

Results.

The target of T14-013 is a unique CD43 epitope.

To more accurately identify the binding epitope of AT14-013, the present inventors generated 11 extracellular truncated Flag-tagged CD43 variants that were expressed in THP1 cells. AT14-013 binding was studied by immunoprecipitation of THP1 cells transduced with these truncated variants. AT14-013 interacts strongly with variants A-F, less with variant F2, less with variant G, and does not interact with variants H-J, as shown in the anti-Flag IP immunoblot (FIG. 16A+B). In FIG. 16B was confirmed by IP AT14-013 using an antibody against the C-terminal region of CD43. All samples contained endogenous CD43, while they contained only truncated CD43, present up to the F2 variant. Thus, the present inventors concluded that the AT14-013 epitope contains at least one amino acid residue that lies between amino acids 133 and 165. Because AT14-013 interacts less with the F2 variant (starting at the amino acid in position 148 as shown in Fig. 13), the inventors also concluded that the AT14-013 epitope contains at least one amino acid residue that is between amino acids 133 and 147.

Example 5 AT14-013 inhibits the growth of AML in vivo.

Known experimental protocols are, for example, described in Miller et al., Blood (2013), vol. 121, no. 5, e1-e4.

In order to evaluate the efficacy of AT14-013 against AML in vivo, immunodeficient mice were treated with reconstituted human hematopoietic cells and inoculated SH-2 cells. Six female NOD.Cg-Prkdc ^scld Il2rg ^1W|SzJ (NSG, The Jackson Laboratory) were humanized by injecting 50,000 CD34+ ^CD38 hematopoietic stem cells into the livers of sublethal neonatal mice (1-5 days old). After 8 weeks, mice were bled to assess engraftment of human hematopoietic cells in their blood. In this experiment, only chimeric mice were used, in which the number of human cells in the peripheral blood was more than 20%. Five out of 6 mice met this criterion and were intravenously inoculated with 10x106 SH-2 cells expressing luciferase and GFP on day 0 (day 0). On day 14, mice were intraperitoneally injected with luciferin (150 mg/kg) and tumor engraftment was assessed by in vivo bioluminescence determination. Based on this measurement, mice were randomized into 2 groups and then dosed intravenously twice a week with AT14-013 or AT10-002 antibody (against the influenza described in WO 2013/081463 as a control) (375 µg). Bioluminescence was measured every week as described above. On day 39, mice were sacrificed by cervical dislocation under deep anesthesia, and the organs were removed and bioluminescence quantified. A single cell suspension was prepared for liver and bone marrow, and the presence of GFP+ SH2 cells was quantified by FACS. Treatment of mice inoculated with SH-2 AML cells resulted in a 90.3% inhibition of tumor growth as measured by whole body measurements of euthanized mice (p<0.001, repeated analysis of variance (ANOVA), (Fig. 17A). in photons per minute (cpm), shows a strong decrease in all organs measured (p=0.0011, duplicate ANOVA, Fig. 17B) This observation is supported by FACS tumor cell counts in bone marrow and liver ( p=0.0017, Duplicate ANOVA, Figure 17C) Therefore, the antibody is specific for the CD43 peptide of the present invention and is particularly useful for in vivo treatment or prevention of a myeloproliferative or lymphoproliferative disorder such as AML.

Example 6

Materials and methods.

Fetal liver, bone marrow and thymic tissue from 16 to 21 weeks of gestation were obtained from mice in a human immune system (HIS) vivarium at AMC (according to Dutch law: Wet Foetaal Weefsel). CD34-enriched tissue mononuclear cell suspensions were prepared by disruption of whole organs using Stomacher equipment followed by density gradient centrifugation and separation of magnetic microcarriers. CD34-enriched fetal bone marrow cell suspension was obtained by density gradient centrifugation and separation of magnetic microcarriers.

Binding of the AT14-013 antibody to cells derived from fetal liver, fetal thymus, and fetal bone marrow was examined by flow cytometry using commercially available antibodies against CD34 (BD, catalog number 343516) and CD38 (BD, catalog number 303522) to distinguish different subsets in given samples.

- 29 040196

Results.

AT14-013 specifically binds to the CD43 oncofetal epitope.

As described above herein, AT14-013 is a CD43-specific antibody that recognizes a unique oncosialylated tumor antigen that is predominantly expressed by AML and MDS blasts. Tumor antigens are either abnormal proteins with tumor-specific expression or aberrantly expressed normal proteins, such as oncofetal antigens, which are antigens that are normally only expressed during ontogeny by fetal tissues. Neoplastic cell transformation is often associated with the expression of oncofetal antigens. The present inventors found that the AT14-013 epitope of CD43 expressed CD34+ ^CD38 hematopoietic stem cells derived from fetal liver and fetal bone marrow, but not CD34+CD38+ progenitor cells or CD34 ^- CD38 mature cells derived from fetal liver and fetal bone marrow (Fig. 18). These results indicate that AT14-013 is able to bind to the oncofetal-sialylated CD43 epitope, which is highly expressed in adult AML and MDS cells.

Example 7 AML blasts from donor 101 (the same donor from which B cells producing AT14-013 were obtained) were stained with AT14-013 and antibodies specific for CD34 and CD38 (same procedure as example 6) and antibodies against CD45 (BD, catalog number 348815) to distinguish the total population of blasts (CD45 dim) from healthy cells in the bone marrow and analyzed by flow cytometry (Fig. 19). This shows that AT14-013 binds the leukemic blasts of the indicated patient in which it was found. AT14-013 binds CD34 ⁺ CD38'blasts, which contain leukemic stem cells.

In this regard, it is concluded that the AT14-013 antibody reacts with autologous leukemic stem cells, making AT14-013 particularly suitable for the treatment or prevention of myeloproliferative or lymphoproliferative disorders, since it also targets leukemic stem cells, which are known to , are more resistant to the action of therapy and are often responsible for the recurrence of the disease after treatment.

It follows that another antibody specific for the CD43 peptide of the present invention, such as an antibody that competes with the AT14-013 antibody for binding to CD43, is also particularly suitable for the treatment or prevention of myeloproliferative or lymphoproliferative disorders.

- 30 040196

Links.

Bennett, J.M. et al., 1976. Proposals for the classification of the acute leukaemias. FrenchAmerican-British (FAB) co-operative group. British Journal of Haematology, 33(4), pp.451-458,

Borche, L. et al., 2005. CD43 monoclonal antibodies recognize the large sialoglycoprotein of human leukocytes. European Journal of Immunology, 17(10), pp 1523-1526

European Patent No. 1974017

Hanly et al. Review of polyclonal antibody production procedures in mammals and poultry. ILAR Journal (1995); Vol.37, No. 3: 93-118

International Patent Application No. WO 2015/093949

International Patent Application No. WO 2006/121240

International Patent Application No. WO 2007/146172

Kim et al. Characterization of two novel mAbs recognizing different epitopes on CD43. Immune Network (2014). Vol. 14, #3: 164-170

Kwakkenbos M.J. Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming. Nat. Med. 2010.16(1):123-8.

de Laurentiis, A. et al., 2011. Mass Spectrometry-Based Identification Of The Tumor Antigen UNI as the Transmembrane CD43 Sialoglycoprotein. Molecular & Cellular Proteomics, 10(5), pp.Ml 11.007898-Ml 11.007898

Malcovati, L. et al., 2013. Diagnosis and treatment of primary myelodysplastic syndromes in adults: recommendations from the European Leukemia Net. Blood, 122(17), pp.2943-2964

Miller et al., Blood(2013), Vol. 121, no. 5, el-e4

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Schmid K, Hediger M.A., Brossmer R, et al. Amino acid sequence of human plasma galactoglycoprotein: identity with the extracellular region of CD43 (sialophorin). Proc. Natl.

Acad. sci. U.S.A. 1992;89(2):663-667

Shelley et al. Molecular characterization of sialophorin (CD43), the lymphocyte surface sialoglycoprotein defective in Wiskott-Aldrich syndrome. Proc. Natl. Acad. sci. U.S.A. 1989; Vol. 86:2819-2823

Swerdlow S.H. WHO classification of Tumors of Haematopoietic and Lymphoid Tissues.

International Agency for Research on Cancer, 2008. ISBN: 978-92-832-2431-0

Tuccillo et al. Cancer-associated CD43 glycoforms as target of immunotherapy. Mol.

Cancer ther. (2014a) 13(3): 752-762

Tuccillo et al. Aberrant glycosylation as a biomarker for cancer: focus on CD43. BioMed research International (2014b) Article ID 742831, 13 pages.

Claims (19)

CLAIM 1. An isolated recombinant or purified CD43 peptide with a length of not more than 100 amino acid residues for the detection and/or production of an immune cell and/or antibody, or its functional part, or its functional equivalent, which are specific to a cell of a myeloproliferative or lymphoproliferative disorder, and the specified peptide contains the amino acid sequence GTITTNSPETSSRTSGAPVTTAASSLETSRGTS, and said peptide has a tumor-specific sialylation profile. 2. MHC complex containing the CD43 peptide according to claim 1. 3. An isolated synthetic or recombinant nucleic acid molecule that encodes a CD43 peptide according to claim 1. 4. The use of the CD43 peptide according to claim 1 for the production, binding, detection and/or production of an immune cell and/or an antibody, or a functional part thereof, or a functional equivalent thereof. 5. The use of the MHC complex according to claim 2 for the production, binding, detection and/or production of an immune cell and/or antibody, or a functional part thereof, or its functional equivalent. 6. The use of a nucleic acid molecule according to claim 3 for the production, binding, detection and/or production of an immune cell and/or an antibody, or a functional part thereof, or a functional equivalent thereof. 7. Use according to any one of claims 4 to 6 for inducing, binding, detecting and/or obtaining an immune cell and/or antibody, or a functional part thereof, or a functional equivalent thereof, which can specifically bind myeloproliferative or lymphoproliferative cells. 8. Use according to claim 7, characterized in that said myeloproliferative cells are acute myeloid leukemia (AML) cells or myelodysplastic syndrome (MDS) cells or chronic myeloid leukemia (CML) cells. 9. Use according to any one of claims 4 to 8, characterized in that said antibody has an activity that induces antibody mediated cellular cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC). 10. An isolated recombinant or purified antibody, or a functional portion thereof, or a functional equivalent thereof, that competes with the AT14-013 antibody for binding to CD43. 11. A diagnostic kit for detecting an immune cell and/or an antibody, wherein said diagnostic kit contains a CD43 peptide according to claim 1 and means for detecting an antibody or immuno - 32 040196 cells. 12. The use of the CD43 peptide according to claim 1 for the diagnosis of AML, MDS or CML. 13. A method for determining whether an individual has AML, said method comprising contacting a CD43 peptide according to claim 1 with antibodies and/or immune cells of said individual and determining binding of said CD43 peptide by at least one of said antibodies and/or immune cells the specified individual. 14. A method for producing an immune cell and/or an antibody that can specifically bind lymphoproliferative cells and/or myeloproliferative cells, preferably an AML-specific immune cell or an AML-specific antibody, said method comprising immunizing a non-human animal with a CD43 peptide according to claim 1 . 15. The method of claim 14, further comprising obtaining from said non-human animal an immune cell and/or an antibody that can specifically bind lymphoproliferative cells and/or myeloproliferative cells, preferably an AML-specific immune cell or an AML-specific antibody. 16. An antibody obtained using the method according to any one of claims 14, 15. 17. Immune cell, which is obtained using the method according to any one of claims 14, 15. 18. A gene delivery vehicle for producing, binding, detecting and/or obtaining an immune cell and/or an antibody, or a functional part thereof, or a functional equivalent thereof, said gene delivery vehicle comprising a nucleic acid molecule according to claim 3. 19. A host cell for producing, binding, detecting and/or obtaining an immune cell and/or an antibody, or a functional part thereof, or a functional equivalent thereof, said host cell comprising the nucleic acid molecule according to claim 3.