NZ738289B2

NZ738289B2 - Aml antigens and uses thereof

Info

Publication number: NZ738289B2
Application number: NZ738289A
Authority: NZ
Inventors: Marijn Aletta Gillissen; Mette Deborah Hazenberg; Martijn Kedde; Wouter Pos; Hergen Spits; Helden Paula Maria Wilhelmina Van
Original assignee: Kling Biotherapeutics Bv
Priority date: 2015-06-24
Filing date: 2016-06-24
Publication date: 2021-11-30

Abstract

The present invention provides novel compounds comprising an antigen of AML cells, and uses thereof.

Description

(12) Granted patent speciﬁcaon (19) NZ (11) 738289 (13) B2 (47) Publicaon date: 2021.12.24 (54) AML ANTIGENS AND USES THEREOF (51) aonal Patent Classiﬁcaon(s): C07K 14/435 (22) Filing date: (73) Owner(s): 2016.06.24 KLING BIOTHERAPEUTICS B.V. (23) te speciﬁcaon ﬁling date: (74) Contact: 2016.06.24 AJ PARK (30) Internaonal Priority Data: (72) Inventor(s): EP 15173662.6 2015.06.24 SPITS, Hergen EP 16150621.7 2016.01.08 GILLISSEN, Marijn Aletta KEDDE, Martijn (86) Internaonal Applicaon No.: HAZENBERG, Mette Deborah VAN HELDEN, Paula Maria Wilhelmina POS, Wouter (87) Internaonal Publicaon : WO/2016/209079 (57) Abstract: The present invenon provides novel compounds sing an angen of AML cells, and uses thereof.

NZ 738289 B2 Title: AML antigens and uses thereof The invention relates to the fields of biology, immunology and medicine.

Acute myeloid leukemia (AML) is a high risk malignancy with five year survival rates of 40 - 50% in patients younger than 60 years of age. For patients over 65 years of age outcomes are even worse, with only less than 20% of ts obtaining durable remissions. Allogeneic stem cell transplantation is frequently applied in the treatment of acute leukemia. It was initially designed to rescue patients from otherwise lethal myeloablative chemotherapy but was subsequently found to be complicated by alloreactive immune response related complications (graft versus host disease; GvHD).

T cell ion of grafts before reinfusion averted GvHD but the observation that T cell depleted graft recipients, r to monozygotic twin donor transplant recipients, experienced much higher rates of relapse made it increasingly clear that the success of allogeneic SCT is dependent on the induction of an anti-leukemic immune response (graft versus ia (GvL)). This has led to the development of strategies to apply allogeneic stem cell transplantation without myeloablative conditioning (reduced intensity stem cell transplantation, RIST), to reduce cytotoxicity and to allow allogeneic SCT in a larger group of patients including older patients and heavily pretreated ts. Preparative regimens in RIST are aimed at decimating the recipients adaptive immune system to prevent graft rejection, t complete ablation of the recipients bone marrow thereby reducing early SCT toxicity. Following transplantation, donor stem cells gradually replace stem cells of the ent and full donor ism is usually achieved within three months after SCT. Although allogeneic SCT is curative in significant s of patients, and much progress has been made in the supportive care of SCT recipients, still 15-30% of patients die as a result of lantation related complications such as GvHD and infectious cations (arising as a result of slow immune recovery following SCT or as a complication of immunosuppressive therapy of GvHD). Hence, although SCT is potentially curative when potent graft versus leukemia (GvL) responses are induced, its eutic success is limited by ost immune responses leading to GvHD which causes high ity and mortality. In view of the high GvHD incidence after allogeneic stem cell transplantation, ing in death of 15- % of the patients, as well as the fact that a suitable donor is not always available for a given patient, alternative treatment approaches would be advantageous. International patent application provides patient-derived, AML-specific, human antibodies that are able to bind intact AML cells. Importantly, the antibodies are derived from human AML patients that received an allogeneic SCT and are in complete remission, demonstrating that the antibodies are effective against AML. The use of antibodies as disclosed in in AML therapy is, therefore, preferred.

Instead of passive immunization with antibodies, immunotherapy would also be an attractive approach. With immunotherapy, a patient suffering from a disease is provided with a disease-specific antigen, which induces and/or enhances an immune response in said patient against the e. Prophylactic or semi-prophylactic applications, wherein an individual is provided with a disease-specific antigen in order to elicit an immune response before onset or before (further) progression of disease, would also be attractive. For instance, immunization with an AML-specific target molecule in order to elicit an immune response against AML would be particularly attractive for patients that received an allogeneic hematopoietic stem cell lantation. Another group for which immunization with such target would be tive is patients suffering from intermediate to high risk ysplastic syndrome (MDS). Such patients have an intermediate to high risk to develop AML, so that it is advantageous to elicit an anti- AML immune response beforehand . The risk of an MDS patient to develop AML is typically ished ing to the international prognostic scoring system (IPSS; see for instance Malcovati et al. 2013). Non-limiting examples of ediate to high risk MDS ts are MDS-RAEB-1 and MDS-RAEB-2 patients. therapy and vaccinations that are specifically ed against AML are currently not available, due to the lack of suitable AML-specific antigens.

AML-specific antigens would also be particularly suitable for determining whether a sample of a patient contains antibodies and/or immune cells able to specifically bind AML cells. Such ation would for instance be valuable for AML diagnosis or for monitoring AML therapy.

It is an object of the t invention to provide novel peptides and compounds comprising an antigen of AML cells; and/or to at least provide the public with a useful choice. Preferably, peptides and compounds are provided that are able to detect and/or elicit an immune response, preferably a specific immune se, against myeloproliferative disorders, more preferably AML.

Summary of the invention In a first aspect the present invention provides an isolated, recombinant or purified CD43 peptide with a length of at most 100 amino acid residues, wherein said peptide comprises an amino acid sequence GTITTNSPETSSRTSGAPVTTAASSLETSRGTS, and n said peptide has an acute myeloid leukemia (AML)-specific sialylation pattern or a myelodysplastic syndrome (MDS)-specific sialylation pattern.

In a second aspect the present invention provides a compound, comprising the CD43 peptide according to the first .

In a third aspect the present invention provides an isolated, synthetic or recombinant nucleic acid molecule encoding the CD43 peptide according to the first aspect.

In a fourth aspect the present ion provides a vector comprising the nucleic acid molecule according to the third aspect.

In a fifth aspect the present ion provides a use of the CD43 peptide according to the first aspect, the nd according to the second aspect, the nucleic acid molecule according to the third aspect, or the vector ing to the fourth aspect, for inducing, isolating, producing, binding, detecting and/or ing an immune cell and/or an antibody, or a functional part thereof that is capable of binding the same n as said antibody, directed against said CD43 peptide, wherein said use is not practiced on a living human being.

In a sixth aspect the present invention provides an in vitro use of the CD43 peptide according to the first aspect, the compound according to the second aspect, the nucleic acid molecule according to the third aspect, or the vector according to the fourth aspect, for inducing, isolating, producing, g, detecting and/or obtaining an immune cell and/or an dy, or a functional part f that is capable of binding the same antigen as said antibody, directed against said CD43 peptide.

In a seventh aspect the present invention provides an isolated, recombinant or purified antibody, or a onal part thereof that is capable of binding the same antigen as said antibody, that es with an antibody comprising a heavy chain CDR1 sequence SPNWWT and a heavy chain CDR2 sequence EIYYGGRVSYNSALRS and a heavy chain CDR3 sequence AGQKNIGCGYSSCFISWFDT and a light chain CDR1 sequence KSSQTILQRSNHLNYLA and a light chain CDR2 sequence WASTRES and a light chain CDR3 sequence PQT for binding to the CD43 peptide according to claim 1 at the GTITTNSPETSSRTSGAPVTTAASSLETSRGTS sequence.

In an eighth aspect the present invention provides an immunogenic composition comprising the CD43 peptide according to the first aspect, or the compound according to the second aspect, or the nucleic acid molecule ing to the third aspect, or the vector ing to the fourth aspect.

In a ninth aspect the present ion provides a diagnostic kit comprising the CD43 peptide according to the first aspect or the compound according to the second aspect, and means for detecting an antibody or immune cell.

In a tenth aspect the present ion provides a use of the CD43 peptide according to the first aspect, or the compound ing to the second , or the nucleic acid molecule according to the third aspect, or the vector according to the fourth aspect, in the preparation of a medicament or prophylactic agent for ng or preventing MDS, AML or CML in a patient in need thereof.

In an eleventh aspect the present ion provides a use of the CD43 peptide according to the first aspect, or a compound according to the second aspect, or the nucleic acid molecule according to the third aspect, or the vector according to the fourth aspect, in the preparation of a ment for treating of a myeloproliferative or lymphoproliferative disorder.

In a twelfth aspect the present invention provides a use of the CD43 peptide according to the first aspect, or the compound according to the second aspect, in the preparation of a diagnostic agent.

In a thirteen aspect the present invention provides a use of a CD43 peptide according to the first aspect, or a compound according to the second aspect, in the preparation of a diagnostic agent for diagnosing AML.

In a fourteenth aspect the present invention es an in vitro method for determining whether an individual has AML, the method comprising contacting the CD43 peptide according to the first aspect, or the nd according to the second , with antibodies and/or immune cells of said individual and determining whether said CD43 peptide or said compound is bound by at least one of said antibodies and/or immune cells of said individual.

In a nth aspect the present invention provides a method for producing an immune cell and/or antibody that is able to specifically bind lymphoproliferative cells and/or myeloproliferative cells, the method comprising immunizing a non-human animal with the CD43 peptide according to the first aspect, or with the compound according to the second aspect, or with the nucleic acid molecule according to the third aspect or with the vector according to the fourth .

In a sixteenth aspect the present invention provides an isolated host cell comprising the nucleic acid molecule according to the third aspect, or the vector according to the fourth aspect.

Detailed description Described herein is an isolated, inant or purified CD43 peptide with a length of at most 100 amino acid residues, wherein said peptide comprises an amino acid sequence with a length of at least 3 amino acid es and at most 51 amino acid residues that is cal to a ce located between CD43 amino acid positions 133 and 184 as depicted Figure 13. Said peptide preferably comprises an amino acid sequence with a length of at least 3 amino acid residues and at most 51 amino acid residues that is identical to a sequence located between CD43 amino acid positions 133 and 183 as depicted Figure 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 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 es, or 51 amino acid residues.

Some embodiments describe an isolated, recombinant or purified CD43 peptide with a length of at most 100 amino acid residues, wherein said peptide comprises an amino acid sequence with a length of at least 3 amino acid residues and at most 33 amino acid residues that is identical to a sequence d between CD43 amino acid positions 133 and 165 as depicted Figure 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 es, or at least 30 amino acid residues, or 33 amino acid residues.

Some embodiments describe an isolated, recombinant or purified CD43 peptide with a length of at most 100 amino acid residues, wherein said peptide comprises an amino acid sequence with a length of at least 3 amino acid es and at most 15 amino acid residues that is identical to a sequence located n CD43 amino acid positions 133 and 147 as depicted Figure 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 discovered that a specific immune response against AML can be detected and/or ed using a CD43 peptide described herein. This finding was unexpected, because CD43 is present on the surface of most kinds of (non-malignant) ytes, as well as on many non-hematopoietic tumor cells like for instance human colon cancer cells, human uterine cervix cancer cells, human lung cancer cells and human breast arcinoma cells. In view of this nt ce of CD43, before the present invention CD43 was not ered an appropriate compound for providing AML icity. Yet, as shown in the es, the AML- specific antibody AT14-013 binds a CD43 peptide described herein. Moreover, antibody AT14-013 binds to various different CD43+ AML cells (figure 2 and 3), but not to CD43+ PBMCs, activated and non-activated T cells, B cells, non-activated monocytes, thymocytes, ALL cells, colon carcinoma cells, non-malignant colon cells, Jurkat cells, Ramos cells or normal bone marrow cells (shown in figure 5). Hence, antibody AT14-013, that is specifically directed against a CD43 peptide as defined in the claims, binds CD43+ AML cells, s it does not bind various other kinds of CD43+ cells.

Interestingly, antibody AT14-013 does not bind different kinds of non-AML, CD43+ hematopoietic stem cells or more mature cells of the lymphoid lineages. It is also shown in the Examples that antibody AT14-013 is able to bind fetal hematopoietic stem cells, from which it is concluded that the CD43 epitope that is recognized by AT14-013 is an oncofetal e. Antibody AT14-013 is also able to bind autologous leukemic stem cells.

Moreover, antibody 13 is able to counteract AML growth in vivo. The present disclosure thus describes a CD43 antigen of AML cells.

CD43, which is also referred to as leukosialin, sialophorin, oglycoprotein, leukocyte sialoglycoprotein or gp115, is a glycosylated mucin-like type I transmembrane protein that is present on the surface of most poietic cells, except erythrocytes.

CD43, encoded by one exon, plays a role in cell-cell interactions. It has a highly glycosylated extracellular region of 235 amino acids. Two CD43 glycoforms have been described, wherein one glycoform mainly contains tetrasaccharides and the other glycoform possesses mainly branched hexasaccharides. Both glycoforms can be expressed on the surface of one cell. CD43 is for instance bed in Shelley et al. (1989) and Schmid et al. (1992). The sequence of human CD43, depicted in Figure 13 is t in the Genbank CCDS se under accession No. CCDS10650.1.

As used herein, the term “CD43 peptide according to the invention” or “CD43 peptide bed herein” refers to a chain of amino acids with a length of at most 100 amino acid residues, wherein said amino acid chain comprises a sequence with a length of at least 3 amino acids residues and at most 51 amino acid residues that is identical to a sequence d between amino acid ons 133 and 184 of a human CD43 protein as depicted in Figure 13, or wherein said amino acid chain comprises a sequence with a length of at least 3 amino acids residues and at most 51 amino acid residues that is identical to a sequence located between amino acid positions 133 and 183 of a human CD43 protein as depicted in Figure 13, or wherein said amino acid chain comprises a sequence with a length of at least 3 amino acids residues and at most 33 amino acid residues that is identical to a sequence located between amino acid ons 133 and 165 of a human CD43 protein as depicted in Figure 13, or wherein said amino acid chain comprises a sequence with a length of at least 3 amino acids residues and at most 15 amino acid residues that is identical to a sequence d between amino acid positions 133 and 147 of a human CD43 protein as depicted in Figure 13.

As explained in detail in the examples, the present disclosure describes the insight that the amino acid sequence between positions 133 and 184 of a human CD43 n comprises an AML epitope that is specifically bound by antibody AT14-013. Said AML epitope comprises one or more amino acid residues that are present n amino acid sequence positions 133 and 165 as depicted in Figure 13. Said AML epitope, which is present on different AML cell lines and AML blasts, and which is not present or exposed on many other CD43+ cells, is therefore ularly suitable for eliciting or detecting an AML-specific immune response. In some embodiments, a CD43 peptide described herein comprises the amino acid sequence GTITTNSPETSSRTS. In some embodiments, a CD43 e described herein comprises the amino acid sequence GTITTNSPETSSRTSGAPVTTAASSLETSRGTS.

In some embodiments, a CD43 peptide described herein comprises the amino acid sequence GTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVS LETSKGTSG.

In some embodiments, a CD43 peptide described herein has a length of at most 90 amino acid residues. In some embodiments, a CD43 peptide described herein has a length of at most 85 amino acid residues or at most 75 amino acid es or at most 70 amino acid residues. In some embodiments, a CD43 peptide described herein has a length of at most 65 amino acid residues or at most 60 amino acid residues or at most 55 amino acid residues or at most 50 amino acid residues or at most 45 amino acid es or at most 40 amino acid residues or at most 35 amino acid residues. In some ments, said CD43 peptide bed herein has a length of at most 52 amino acid residues, or at most 51 amino acid residues, or at most 33 amino acid residues, or at most 30 amino acid residues or at most 25 amino acid residues or at most 20 amino acid residues or at most 15 amino acid residues. In some embodiments, a CD43 peptide described herein has a length of 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.

In some embodiments, said CD43 e described herein has a length of at least 52 amino acid es or at least 51 amino acid residues, wherein said peptide comprises an amino acid sequence that is identical to a sequence located n amino acid positions 133 and 184 of a human CD43 protein as depicted in Figure 13. In some embodiments, said CD43 peptide described herein has a length of at least 51 amino acid residues, wherein said amino acid es are identical to the amino acids between amino acid positions 133 - 183 of a human CD43 protein as depicted in Figure 13.

In some embodiments, a CD43 peptide described herein has a length of at least 33 amino acid residues and comprises an amino acid sequence that is cal to the sequence located between amino acid positions 133 and 165 of the human CD43 protein as depicted in Figure 13.

In some embodiments, a CD43 peptide described herein has a length of at least 15 amino acid residues and comprises an amino acid sequence that is identical to the sequence located between amino acid positions 133 and 147 of the human CD43 protein as depicted in Figure 13.

In some ments, a CD43 peptide described herein consists of the sequence GTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVSLETSK GTSG.

In some embodiments, a CD43 peptide described herein ts of the sequence GTITTNSPETSSRTSGAPVTTAASSLETSRGTS.

In some embodiments, a CD43 peptide described herein consists of the sequence GTITTNSPETSSRTS.

As used herein, the expressions “sequence located between CD43 amino acid positions X and Y as depicted in Figure 13”, “sequence located between amino acid positions X and Y of the human CD43 protein as ed in Figure 13”, “wherein said amino acid residues are identical to the amino acids between amino acid positions X - Y of a human CD43 protein as depicted in Figure 13” and “an amino acid sequence that is identical to the sequence located between amino acid positions X and Y of the human CD43 protein as depicted in Figure 13” encompass sequences that are located between the recited positions and that include the amino acid(s) of position X and/or Y. In addition, the terms embrace sequences that are located between the recited positions and that do not contain the amino acid(s) of positions X and/or Y. In other words, in some ments the amino ) of the recited positions X and/or Y are t in a CD43 peptide described herein, whereas in other embodiments the amino acids of the d positions X and/or Y are absent.

Besides the recited amino acid sequences that are identical to a sequence located between amino acid positions 133 and 184, or to a sequence located between amino acid positions 133 and 183, or to a sequence located between amino acid positions 133 and 165, or to a sequence located n amino acid positions 133 and 147, of a human CD43 protein as depicted in Figure 13, a CD43 peptide described herein may further comprise other amino acid residues. In some embodiments, said other amino acid residues are not derived from a CD43 sequence. Said other amino acid residues, which are also referred to herein as “non-CD43 amino acid residues” may for instance function to enhance stability, and/or to e immunogenicity, and/or to couple the CD43 peptide to another moiety such as for instance a molecular scaffold or carrier. Nonlimiting examples of such scaffold or carriers are keyhole limpet anin and CLIPS scaffolds (such as for instance bis(bromomethyl)benzene, tris(bromomethyl)benzene and tetra(bromomethyl)benzene, described in ). Some embodiments therefore describe an ed, recombinant or ed peptide with a length of at most 100 amino acid es, wherein said peptide comprises an amino acid ce with a length of at least 3 amino acid residues and at most 52 amino acid residues, or at most 51 amino acid residues, that is identical to a sequence located n CD43 amino acid positions 133 and 184 as depicted Figure 13, and wherein said peptide also comprises 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 is not present in human CD43 as depicted in Figure 13.

Some ments describe an isolated, recombinant or purified peptide with a length of at most 100 amino acid residues, wherein said peptide comprises an amino acid sequence with a length of at least 3 amino acid residues and at most 51 amino acid residues that is identical to a sequence located between CD43 amino acid positions 133 and 183 as depicted Figure 13, and wherein said peptide also comprises 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 is not t in human CD43 as depicted in Figure 13. In some embodiments, the length of said amino acid ce is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid es, 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 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.

Some embodiments describe an isolated, recombinant or purified peptide with a length of at most 100 amino acid residues, wherein said peptide comprises an amino acid sequence with a length of at least 3 amino acid residues and at most 33 amino acid residues that is identical to a sequence located between CD43 amino acid positions 133 and 165 as depicted Figure 13, and wherein said peptide also comprises 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 , 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 is not present in human CD43 as depicted in Figure 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 es, 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 residues.

Some ments describe an isolated, inant or purified peptide with a length of at most 100 amino acid residues, wherein said peptide comprises an amino acid sequence with a length of at least 3 amino acid residues and at most 15 amino acid residues that is identical to a sequence located between CD43 amino acid positions 133 and 147 as depicted Figure 13, and wherein said peptide also comprises 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 , or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, non-CD43 amino acid es, wherein the full length ce of said non-CD43 amino acid es is not t in human CD43 as depicted in Figure 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 above mentioned es are also ed by the term “CD43 peptide according to the present invention” or the term “CD43 peptide described herein”.

Some embodiments describe a compound comprising a CD43 peptide described . Some embodiments describe an immunogenic compound comprising a CD43 e described herein. In some embodiments said CD43 peptide is coupled to a pharmaceutically acceptable r or scaffold.

In some embodiments, a CD43 e described herein is a truncated CD43 molecule with a length of at most 100 amino acid residues. Preferably, said truncated CD43 molecule is devoid of the intracellular region of a wild type human CD43. In preferred embodiments, said truncated CD43 molecule is devoid of both the intracellular region and the transmembrane region of a wild type human CD43. In further preferred embodiments, said CD43 peptide described herein is a truncated CD43 extracellular region with a length 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 es, or at most 52 amino acid residues, or at most 51 amino acid residues, or at most 50 amino acid residues, or at most 45 amino acid es, or at most 40 amino acid residues, or at most 35 amino acid es, or at most 33 amino acid residues, or at most 30 amino acid residues, or at most 25 amino acid residues, or at most 20 amino acid residues, that comprises an amino acid sequence with a length of at least 3 amino acid residues and at most 52 amino acid residues or at most 51 amino acid residues that is identical to a sequence located between CD43 amino acid positions 133 and 184 as depicted Figure 13.

In some embodiments, the length of said amino acid ce 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 ments, said CD43 peptide described herein is a truncated CD43 extracellular region with a length 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 at most 50 amino acid residues, or at most 45 amino acid residues, or at most 40 amino acid residues, or at most 35 amino acid residues, or at most 33 amino acid residues, or at most 30 amino acid residues, or at most 25 amino acid residues, or at most 20 amino acid residues, that comprises an amino acid sequence with a length of at least 3 amino acid residues and at most 51 amino acid es that is identical to a sequence located between CD43 amino acid positions 133 and 183 as depicted Figure 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 described herein is a truncated CD43 extracellular region with a length 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 at most 50 amino acid residues, or at most 45 amino acid es, or at most 40 amino acid residues, or at most 35 amino acid residues, or at most 33 amino acid residues, or at most 30 amino acid residues, or at most 25 amino acid residues, or at most 20 amino acid residues, that comprises an amino acid ce with a length of at least 3 amino acid residues and at most 33 amino acid es that is identical to a sequence located between CD43 amino acid positions 133 and 165 as depicted Figure 13. In some embodiments, the length of said amino acid ce 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 es, or at least 20 amino acid residues, or at least 25 amino acid residues or at least amino acid residues or 33 amino acid residues.

In some embodiments, said CD43 peptide described herein is a truncated CD43 extracellular region with a length 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 at most 50 amino acid residues, or at most 45 amino acid residues, or at most 40 amino acid residues, or at most 35 amino acid residues, or at most 33 amino acid residues, or at most 30 amino acid es, or at most 25 amino acid residues, or at most 20 amino acid es, that comprises an amino acid sequence with a length of at least 3 amino acid residues and at most 15 amino acid residues that is cal to a sequence located between CD43 amino acid positions 133 and 147 as depicted Figure 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 es, 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.

Some embodiments describe a CD43 peptide described herein that is a truncated CD43 extracellular region with a length 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 52 amino acid es, or at most 51 amino acid es, or at most 50 amino acid residues, or at most 45 amino acid residues, or at most 40 amino acid residues, or at most 35 amino acid residues, or at most 33 amino acid residues, or at most 30 amino acid residues, or at most 25 amino acid residues, or at most 20 amino acid residues, or at most 15 amino acid residues.

As is known to the skilled person, once an immunogenic sequence has been provided, it has become possible to alter the sequence to some extent, thereby preferably optimizing the genicity and/or stability of the resulting immunogen. This is for instance done by mutagenesis procedures where after the stability and/or immunogenicity of the resulting nds are preferably tested and an improved AML- specific antigenic compound is selected. A skilled person is well capable of generating antigen variants starting from a certain amino acid sequence. For instance, conservative amino acid tution is applied. es of conservative amino acid substitution include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue, and the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine. In some ments, a replacement net analysis is d out, which involves replacement of one or more amino acid residues by any other amino acid residue, and testing the resulting compounds.

Some embodiments therefore describe an isolated, recombinant or purified CD43 peptide with a length of at most 100 amino acid residues that comprises, or essentially consists of, an amino acid sequence having a length of at least 3 amino acid es and at most 52 amino acid residues or at most 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 with a sequence located between amino acid ons 133 and 184 of the human CD43 n as depicted in Figure 13. Some embodiments be an isolated, recombinant or purified CD43 peptide that comprises an amino acid sequence having a length of 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 with the sequence located between amino acid positions 133 and 183 of the human CD43 protein as depicted in Figure 13.

Some embodiments describe an ed, recombinant or purified CD43 peptide with a length of at most 100 amino acid es that comprises, or essentially consists of, an amino acid sequence having a length of 33 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 ably at least 97%, more preferably at least 98%, more preferably at least 99% ce identity with the sequence located between amino acid positions 133 and 165 of the human CD43 protein as depicted in Figure 13.

Some embodiments describe an isolated, recombinant or purified CD43 peptide with a length of at most 100 amino acid residues that comprises, or essentially consists of, an amino acid sequence having a length 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 ty with the sequence located between amino acid positions 133 and 147 of the human CD43 n as depicted in Figure 13.

The term “% ce identity” is defined herein as the percentage of residues in a candidate amino acid sequence that is identical with the es in a nce ce after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. s and computer programs for the alignment are well known in the art. One computer program which may be used or adapted for purposes of determining whether a candidate sequence falls within this definition is "Align 2", authored by Genentech, Inc., which was filed with user documentation in the United States Copyright Office, Washington, D.C. 20559, on Dec. 10, 1991.

An isolated, recombinant or purified CD43 peptide as defined herein is also referred to as “a CD43 peptide ing to the invention” or “a CD43 antigen according to the invention” or a “CD43 peptide described herein”. In some embodiments, the amino acid residues of a CD43 peptide described herein are chosen from the 20 amino acid residues that lly occur in eukaryotes, which are also referred to as “standard” or “canonical” amino acids. Alternatively, non-natural amino acid residues are included in a CD43 peptide described herein, such as for instance D-amino acids (i.e. D-stereoisomers of amino acids) or N-methyl amino acids.

A CD43 peptide described herein is preferably glycosylated. Such peptide more accurately reflects the natural AML antigen in vivo, in view of the fact that the l CD43 n on the cell surface is heavily glycosylated. In some preferred embodiments, said CD43 peptide described herein comprises sialic acid residues (also called α-N-acetylneuraminic acid). In vivo, human CD43 is highly sialylated. Treatment with neuraminidase s sialic acid residues from the CD43 glycoprotein. In some ments, ore, a CD43 peptide described herein is neuraminidase-sensitive.

Some ments relate to a CD43 peptide described herein that has been oncosialylated , meaning that said CD43 peptide has a tumor-specific sialylation pattern. As used herein, a CD43 peptide described herein with a “tumor-specific sialylation pattern” encompasses a CD43 peptide described herein with a sialylation pattern that has been produced by a tumor cell, or a CD43 peptide described herein with a sialylation pattern that is identical to, or at least 90% or at least 95% or at least 97% similar to, a sialylation pattern as produced by a tumor cell.

Some ments describe a CD43 peptide described herein that has been produced by an AML cell. Some ments describe a CD43 peptide described herein that has been produced by a cell of a cell line derived from an AML cell. Such CD43 peptide will have a glycosylation pattern that very closely resembles the glycosylation pattern of AML cells in an AML patient in vivo. In some embodiments, said CD43 peptide described herein has been produced by a THP-1 cell. In some embodiments, said CD43 peptide bed herein has been produced by a Kasumi 3 cell, or by an HL60 cell, or by a KG1a cell, or by an SH2 cell, or by a MonoMac6 cell, or by a Molm13 cell, or by a CML K562 cell.

As used , a CD43 e described herein having a glycosylation pattern that is similar or identical to a glycosylation pattern that results from expression of said CD43 peptide in an AML cell, for instance in an AML blast or in an AML cell line, is referred to as a CD43 peptide described herein having an AML-specific glycosylation pattern. Some embodiments thus be a CD43 peptide described herein that has an AML-specific glycosylation pattern.

Some embodiments describe a CD43 peptide described herein that has been produced by an MDS cell. Some embodiments describe a CD43 e described herein that has been produced by a cell of a cell line derived from an MDS cell. Such CD43 peptide will have a glycosylation pattern that very closely les the glycosylation pattern of MDS cells in an MDS patient in vivo.

Some ments describe CD43 peptides described herein that have an MDS- specific glycosylation pattern. These es have a glycosylation pattern that is similar or identical to a glycosylation pattern that results from expression of a CD43 peptide described herein in an MDS cell, for instance in an MDS blast or in an MDS cell line.

In other embodiments, a CD43 peptide described herein is bed that has been produced by a host cell, using in vitro glycoengineering (for instance according to Roche Diagnostics GmbH). According to these embodiments, host cells are described with the enzymes alpha-2,6-Sialyltransferase and/or 2,3-Sialyltransferase, so that upon production of a CD43 peptide described herein, the peptide will be sialylated. r described is therefore a CD43 e described herein that has been produced by a cell that contains alpha-2,6-Sialyltransferase and/or 2,3-Sialyltransferase. In some embodiments, said alpha-2,6-Sialyltransferase and/or alpha-2,3-Sialyltransferase comprises exogenous alpha-2,6-Sialyltransferase and/or ous alpha-2,3- Sialyltransferase, meaning that the enzyme has been introduced recombinantly into the host cell (or into a parent host cell from which the current host cell originates). Some embodiments describe a CD43 peptide described herein that has been ed by a host cell that contains an exogenous nucleic acid sequence encoding alpha-2,6- Sialyltransferase and/or alpha-2,3-Sialyltransferase.

Anti-CD43 antibodies are known in the art. However, it is clear that these antibodies recognize a different e. For instance, as shown in Table 1 of Kim et al, 2014, anti-CD43 monoclonal antibodies (mAbs) YG5, 2C8, 8E10 and DFT-1 do bind AML cells, but also many other non-AML cells, including CEM7, Jurkat, IM9, Ramos, Raji, Daudi, Reh, normal bone marrow and PBL cells, are bound by some or all of these known antibodies (Kim et al, 2014). Hence, antibodies YG5, 2C8, 8E10 and DFT-1 are not at all specific for AML. Contrary, antibody AT14-013, that specifically binds the CD43 peptides described herein, does not bind Jurkat cells, Ramos cells, normal bone marrow cells or PBL cells/PBMCs (figure 5 and 9b). Antibodies YG5, 2C8, 8E10 and DFT-1 thus bind another CD43 epitope. dy UN1 (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 was concluded that the protein core of this epitope includes CD43 amino acids 64 to 83. This antigen is expressed by human thymocytes, by the leukemic cell lines HPB-ALL, H9 and Molt-4, and in a subpopulation of peripheral blood CD4+ hocytes. Antibody AT14-013, however, does not bind human thymocytes, indicating that a different AML antigen is described herein.

International patent application describes antibodies 5F1, 51-41 and 138-10, which recognize CD43 present on the surface of the human colorectal arcinoma cell line 5 and the human gastric carcinoma cell line NCI-N87.

AML is not mentioned in . Antibody AT14-013, that specifically binds the AML-specific CD43 peptides described herein, does not bind Colo205, as shown in the Examples. Antibodies 5F1, 51-41 and 138-10 thus bind a different CD43 epitope.

International patent ation describes antibodies EB-1, EB-2 and EB-3 that are able to recognize an unglycosylated region of CD43, consisting of CD43 amino acids 73-81. This n is present on thymocytes, on some hematopoietic precursors in bone marrow, on AML cells, on acute lymphogenous leukemia (ALL) cells and on c myelogenous leukemia (CML) cells. EB-1 recognizes its antigen in both sialidase-treated and ted CD43 molecules. Contrary, glycosylated CD43 peptides described herein are no longer recognized by antibody AT14-013 after treatment with sialidase (neuramidase), meaning that the present disclosure describes an AML n that is neuramidase-sensitive, contrary to the antigen of EB-1, EB-2 and EB-3. rmore, antibody 13 does not bind ALL cells or thymocytes, contrary to antibodies EB-1, EB-2 and EB-3. Moreover, the CD43 es described herein comprise an amino acid sequence with a length of at least 3 amino acid residues and at most 51 amino acid es that is identical to a sequence located within amino acid residues 133 and 184 of CD43, which domain is different from CD43 amino acid positions 73-81 that are recognized by antibodies EB-1, EB-2 and EB-3. Antibodies EB-1, EB-2 and EB-3 thus also bind another CD43 epitope.

In conclusion, a novel AML-specific antigen is described in the present disclosure.

A CD43 peptide described herein preferably has a length of at most 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 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 ments, said length is at least 20 amino acid residues. In some embodiments, said length is at least 25 amino acid residues. In some embodiments, said length is at least 30 amino acid residues. In some embodiments, said length is at least 33 amino acid residues. In some embodiments, said length is at least 35 amino acid residues. In some embodiments, said length is at least 40 amino acid residues. In some embodiments, said length is at least 45 amino acid residues. In some embodiments, said length is at least 50 amino acids. In some embodiments, said length is at least 51 amino acids. In some embodiments, said length is at least 52 amino acids.

In some ments, said length is at most 90 amino acid residues, or at most 85 amino acid residues or at most 75 amino acid residues or at most 70 amino acid es or at most 65 amino acid residues or at most 60 amino acid residues or at most 55 amino acid residues or at most 52 amino acid residues or at most 51 amino acid residues or at most 50 amino acid residues or at most 45 amino acid residues or at most 40 amino acid residues or at most 35 amino acid residues or at most 30 amino acid residues or at most 25 amino acid residues or at most 20 amino acid. In some embodiments, said peptide has a length of at most 52 amino acid residues and at least 3 amino acid es, preferably at least 5 amino acid residues, or at least 6 amino acid residues, or at least 7 amino acid residues, or at least 8 amino acid residues, or at least 9 amino acid residues, or at least 10 amino acid residues. In some embodiments, said peptide has a length of 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 8 amino acid residues, or at least 9 amino acid es, or at least 10 amino acid residues. In some embodiments, said peptide has a length of at most 33 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 8 amino acid residues, or at least 9 amino acid residues, or at least amino acid residues. In some embodiments, said peptide has a length of at most 15 amino acid es 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 8 amino acid es, or at least 9 amino acid residues, or at least 10 amino acid residues.

In some embodiments, a CD43 e described herein consists of an amino acid sequence with a length of at least 3 amino acid residues and at most 51 amino acid residues or at most 51 amino acid residues that is identical to a sequence located n CD43 amino acid positions 133 and 183 as depicted Figure 13. In some embodiments, a CD43 peptide described herein consists of an amino acid sequence with a length of at least 5 amino acid residues and at most 40 amino acid residues that is identical to a sequence located between CD43 amino acid positions 133 and 183 as depicted Figure 13. In some embodiments, a CD43 peptide described herein consists of an amino acid sequence with a length of at least 5 amino acid residues and at most 33 amino acid residues that is identical to a sequence located between CD43 amino acid positions 133 and 183 as depicted Figure 13. In some embodiments, a CD43 peptide described herein consists of an amino acid sequence with a length of at least 5 amino acid residues and at most 20 amino acid residues that is identical to a sequence located between CD43 amino acid positions 133 and 183 as ed Figure 13.

In some embodiments, a CD43 peptide described herein consists of an amino acid sequence with a length of at least 5 amino acid residues and at most 33 amino acid residues that is identical to a sequence located between CD43 amino acid positions 133 and 165 as depicted Figure 13. In some embodiments, a CD43 peptide described herein consists of an amino acid sequence with a length of at least 5 amino acid es and at most 30 amino acid residues that is identical to a ce located between CD43 amino acid ons 133 and 165 as ed Figure 13. In some ments, a CD43 e bed herein consists of an amino acid sequence with a length of at least 5 amino acid residues and at most 20 amino acid residues that is identical to a sequence located between CD43 amino acid positions 133 and 165 as depicted Figure 13.

In some embodiments, a CD43 peptide described herein consists of an amino acid sequence with a length of at least 5 amino acid residues and at most 15 amino acid es that is identical to a sequence located between CD43 amino acid positions 133 and 147 as depicted Figure 13. In some embodiments, a CD43 peptide described herein consists of an amino acid ce with a length of at least 8 amino acid es and at most 15 amino acid residues that is identical to a sequence located between CD43 amino acid positions 133 and 147 as depicted Figure 13. In some embodiments, a CD43 peptide described herein consists of an amino acid sequence with a length of at least 10 amino acid residues and at most 15 amino acid residues that is identical to a sequence located n CD43 amino acid positions 133 and 147 as depicted Figure 13.

In some embodiments, the above mentioned peptides are glycosylated, preferably comprising sialic acid residues, in order to better mimic the natural AML-specific antigen. In some embodiments, the above mentioned peptides are onco-sialylated. In some embodiments, the above mentioned es have been produced by AML cells or an AML cell line, preferably THP-1 cells. In some embodiments, the above mentioned peptides have been produced by MDS cells or an MDS cell line.

Nucleic acid molecules, or functional equivalents f, encoding a CD43 peptide described herein are also encompassed by the present disclosure. Further described is therefore an isolated, synthetic or recombinant c acid molecule, or a functional equivalent thereof, ng a CD43 peptide described herein. As used , a c acid molecule or nucleic acid ce of the invention or as described herein preferably comprises a chain of nucleotides, more preferably DNA, cDNA or RNA. In other embodiments a nucleic acid molecule or nucleic acid sequence described herien comprises other kinds of nucleic acid structures such as for instance a DNA/RNA helix, peptide nucleic acid (PNA), locked nucleic acid (LNA) and/or a me. Such other nucleic acid structures are referred to as functional equivalents of a nucleic acid sequence. The term “functional equivalent of a nucleic acid molecule” thus encompasses a chain sing non-natural nucleotides, ed nucleotides and/or non-nucleotide building blocks which t the same function as natural tides.

Some embodiments describe a nucleic acid molecule or functional equivalent thereof described herein, wherein a human nucleic acid ce has been codon optimized for a non-human cell, for instance for a non-human producer cell like E.coli, a Chinese hamster ovary (CHO) cell, an NSO cell (which is a mouse myeloma) or a 293(T) cell. This means that one or more codons from said human nucleic acid sequence ve been replaced by one or more codons that are preferred by said non-human cell.

As used herein, an isolated, synthetic or recombinant nucleic acid molecule, or a functional equivalent thereof, encoding a CD43 peptide described herein is also referred to as “a nucleic acid molecule or functional equivalent according to the invention”.

Now that the disclosure has described CD43 peptides comprising a novel AML- specific antigen, many applications have become possible. For instance, in some embodiments a CD43 peptide described herein is used for inducing, isolating and/or obtaining immune cells and/or antibodies, or functional parts or functional derivatives thereof, that are able to specifically bind proliferative and/or myeloproliferative cells. Immune cells and/or antibodies, or onal parts or functional derivatives thereof, that are induced, isolated and/or obtained with a CD43 peptide described herein are particularly le for ent or prevention of myeloproliferative or lymphoproliferative disorders. Even more so in view of the fact that antibody AT14-013, which is an dy that is specific for a CD43 peptide described herein, also targets ic stem cells, which are known to be more therapy resistant and often responsible for relapse of disease after treatment. In some embodiments a CD43 peptide bed herein is used for inducing and/or obtaining AML-specific immune cells and/or AML- specific antibodies. For instance, a non-human animal is immunized with one or more CD43 peptides described herein, or with an immunogenic compound comprising a CD43 e described herein, or with a nucleic acid molecule or functional equivalent thereof encoding a CD43 peptide bed herein, or with a vector comprising a nucleic acid molecule or functional lent described herein, preferably followed by one or more booster administrations. Subsequently, immune cells and/or dies that are specific for lymphoproliferative and/or myeloproliferative cells, preferably specific for AML, are harvested from said non-human animal. In some embodiments, said immune cells se T cells, such as for instance NK cells or T-helper cells.

In some embodiments, said immune cells harvested from said immunized nonhuman animal comprise B cells. for instance, AML-specific B-cells are particularly suitable for the production of AML-specific dies. AML-specific B-cells harvested from said immunized animal are for instance used for the production of hybridomas, from which AML-specific dies 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 as for instance described in European Patent No. 1974017 and US patent US 9,127,251. This way, long term replicating B cell cultures are generated, wherein the B cells both replicate and produce antibody. In some embodiments, AML-specific antibodies ed by said hybridomas or by such B cell culture are ted and for instance used for anti-AML therapy, preferably after zation of the antibodies in order to reduce side-effects. In some embodiments, an antibody and/or B cell obtained from said non-human animal is tested for competition with antibody AT14-013 for g to CD43. This is for instance done by incubating AML cells with said antibody or B cell obtained from said non-human animal, and subsequently adding antibody AT14-013. As a control, AML cells are preferably incubated with antibody AT14-013 in the absence of any other antibody or B cell. If preincubation of AML cells with an antibody or B cell obtained from said non-human animal appears to affect the binding of AT14-013 to said AML cells, it is concluded that said dy or B cell obtained from said non-human animal competes with antibody AT14-013 for binding to CD43.

In some embodiments, the le domain-encoding nucleic acid sequences of B cells from said non-human animal are sequenced in order to obtain the nucleic acid sequences of AML-specific variable domains, where after one or more nucleic acid molecules comprising these sequences are introduced in producer cells, such as for instance , Chinese hamster ovary (CHO) cells, NSO cells or 293(T) cells, for the production of AML-specific antibodies. Said one or more nucleic acid sequences are preferably codon optimized for said producer cell. As used herein, the term “codon” means a triplet of nucleotides (or functional equivalents f) that encode a specific amino acid residue. The term “codon optimized” means that one or more codons from the original, animal nucleic acid ce are replaced by one or more codons that are preferred by a cell from another species, such as for instance a certain producer cell.

These replacement codons preferably encode the same amino acid residue as the original animal codon that has been replaced.

In some embodiments, CD43-specific antibodies obtained from said non-human animal or from immune cells of said non-human animal are humanized, meaning that at least part of the animal amino acid sequence, preferably at least part or the whole of the framework sequences, is replaced by a human sequence in order to reduce adverse sideeffects in .

Animal immunization protocols, including suitable administration procedures and adjuvants, procedures for obtaining and purifying antibodies and/or immune cells from such immunized animals, competition experiments and humanization procedures of non- human antibodies are well known in the art. Reference is for instance made to Hanly et al, 1995.

In some embodiments, a CD43 peptide described herein, or a nd comprising a CD43 e described herein, is used for screening a phage display library in order to identify and/or isolate AML-specific immunoglobulins (typically Fab fragments). In some embodiments, a naïve phage display library is used. In preferred embodiments, a phage display library derived from one or more AML ts is used, so that the library will y be biased towards AML. In some ments, an AML- specific immunoglobulin obtained from said phage display y is tested for competition with antibody AT14-013 for binding to CD43. This is for instance done using a competition test described herein.

Further described is therefore a use of a CD43 peptide described herein, or a use of an genic compound described herein, or a use of a nucleic acid molecule or functional lent thereof encoding a CD43 peptide described , or a use of a vector comprising a nucleic acid le or functional equivalent described herein, for inducing, isolating and/or obtaining an immune cell or an dy, or a functional part or functional equivalent f, such as for instance a Fab fragment. Said immune cell or antibody or functional part or functional equivalent thereof is preferably able to specifically bind lymphoproliferative cells and/or roliferative cells. Preferably, said myeloproliferative cells are AML cells, MDS cells and/or CML cells, most preferably AML cells. In some embodiments, a CD43 peptide described herein or an immunogenic compound described herein or a c acid molecule or functional equivalent bed herein or a vector comprising a nucleic acid molecule or functional equivalent described herein is used for inducing and/or obtaining an antibody that is able to induce antibodydependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). A non-limiting example of such antibody is AT14-013, as shown in the Examples. The fact that a CD43 peptide described herein, or an immunogenic compound described herein, is able to induce and/or obtain an antibody with ADCC and/or CDC inducing activity means that antibodies can be induced or obtained that are functional in vivo.

A CD43 peptide or compound described herein for use as an immunogen is also herewith described, as well as a nucleic acid molecule or functional equivalent described herein, or a vector comprising a nucleic acid molecule or functional equivalent described herein, for use as an gen.

Some embodiments describe a method for producing immune cells and/or antibodies that are able to specifically bind lymphoproliferative cells and/or myeloproliferative cells, such as for instance an AML-specific immune cell or an AML- specific antibody, the method comprising immunizing a non-human animal with a CD43 peptide described herein, or with a compound described herein or with a nucleic acid molecule or functional lent described herein or with a vector comprising a nucleic acid molecule or functional lent described . The method preferably further comprises harvesting an immune cell and/or antibody that is able to specifically bind lymphoproliferative cells and/or myeloproliferative cells from said non-human . In some embodiments, an AML-specific immune cell and/or an AML-specific antibody is harvested from said non-human animal. In some embodiments, a B cell and/or dy obtained from said non-human animal is tested for competition with antibody AT14-013 for binding to CD43. An immune cell and/or antibody that is able to specifically bind lymphoproliferative cells and/or roliferative cells obtainable by a method described herein is also described th. Some embodiments describe an AML- ic antibody or an AML-specific immune cell obtainable by a method described herein for producing an AML-specific immune cell or an AML-specific antibody. Such AML-specific antibody preferably es with antibody AT14-013 for binding to CD43.

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

In view of the fact that antibody AT14-013 ically binds a CD43 peptide described herein, other dies that are obtained, produced or selected with a CD43 peptide described herein will typically e with antibody AT14-013 for binding to CD43. Contrary, current CD43 antibodies that are known in the art do not compete with antibody AT14-013, whereas these known antibodies do compete with each other, as shown in Figure 9. These other antibodies known in the art thus clearly bind an epitope that is ent from the epitope of AT14-013. Further described is, therefore, an ed, recombinant or purified antibody, or a functional part or a functional equivalent thereof, that competes with antibody AT14-013 for binding to CD43. Said isolated, recombinant or purified antibody, or functional part or functional equivalent thereof, preferably competes with antibody AT14-013 for g to at least part of an epitope that is located between amino acids 133 and 184 of a CD43 sequence as depicted in Figure 13. Said isolated, recombinant or purified antibody or functional part or functional equivalent typically es with antibody AT14-013 for binding to a CD43 peptide described herein.

The term “antibody” as used herein, refers to an immunoglobulin protein comprising two heavy chains, bound to each other, wherein each heavy chain is also paired with a light chain.

A “functional part of an antibody” is defined herein as a part that has at least one shared property as said antibody in kind, not necessarily in amount. Said functional part is capable of binding the same antigen as said antibody, albeit not necessarily to the same extent. A functional part of an antibody preferably comprises at least a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, a functional part of an dy comprises at least a heavy chain variable domain (VH). Non-limiting examples of a onal part of an antibody are a single domain antibody, a single chain antibody, a nanobody, an unibody, a single chain variable fragment , a bi-specific T-cell r (BiTE), a Fab fragment and a F(ab')2 fragment.

A “functional equivalent of an antibody” is defined herein as an artificial binding compound, comprising at least one CDR sequence of an antibody, ably a heavy chain CDR3 sequence. Said functional equivalent preferably comprises the heavy chain CDR3 sequence of an antibody, as well as the light chain CDR3 sequence of said antibody. More preferably, said functional equivalent ses the heavy chain CDR1, CDR2 and CDR3 sequences of an antibody, as well as the light chain CDR1, CDR2 and CDR3 sequences of said antibody. A onal equivalent of an antibody is for instance produced by altering an antibody such that at least an antigen-binding property of the resulting nd is essentially the same in kind, not necessarily in amount. This is done in many ways, for ce through conservative amino acid substitution, whereby an amino acid residue is substituted by another residue with generally similar properties (size, hobicity, etc), such that the overall functioning of the antibody is essentially not affected. A non-limiting example of a functional equivalent of an antibody is an antibody with a modified Fc tail, which Fc tail has for ce been modified by amino acid replacement(s) and or glycosylation alteration(s).

As is well known by the skilled person, a heavy chain of an antibody is the larger of the two types of chains making up an immunoglobulin molecule. A heavy chain comprises a constant domain and a variable domain, which variable domain is involved in antigen g. A light chain of an antibody is the smaller of the two types of chains making up an immunoglobulin molecule. A light chain comprises a constant domain and a le . The variable domain is often, but not always, together with the variable domain of the heavy chain involved in antigen binding.

Complementary-determining regions (CDRs) are the hypervariable regions present in heavy chain variable domains and light chain variable domains. In case of whole antibodies, the CDRs 1-3 of a heavy chain and the CDRs 1-3 of the connected light chain together form the antigen-binding site.

As used herein, an immune cell, antibody or functional part or onal equivalent thereof is “specific” for AML if it is able to bind AML cells with a binding affinity that is at least two times higher than the binding affinity of an irrelevant control antibody or control immune cell to said AML cells (wherein the control antibody or control immune cell is not specific for said AML cells). For instance, AML binding by 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 variable domain of the dy or BCR or TCR allow these two structures to bind together (an interaction similar to a lock and key), as opposed to random, non-specific sticking of antibodies or BCRs or TCRs. Some reactivity towards other types of cells is, however, embraced within the term “AML-specific”. As an antibody or BCR or TCR typically recognizes an epitope of an antigen, and as such e might be t on other cells as well, AML-specific antibodies, B cells or T cells might then recognize such other cells. Non-limiting examples of such other cells are MDS and CML cells. Also melanoma cells and cytes appear to be bound by the ecific antibody AT14-013, albeit to a lesser extent. Hence, the term “AML-specific” does not e binding of the antibodies or B cells or T cells to another cell that ns at least part of the same epitope. It is, however, shown in the Examples that many CD43+ cells, including CD43+ hematopoietic cells such as for instance PBMCs, (precursor) T cells and B cells, do not contain an AML antigen as described by the present disclosure.

CD43 peptides described herein are particularly suitable for testing for the presence of AML-specific binding compounds, such as for instance AML-specific antibodies or AML-specific immune cells such as B cells or T cells, in a biological sample.

For instance, a sample from an individual, or a fraction of such sample that comprises antibodies, B cells and/or T cells, is incubated with a CD43 peptide described herein, or with a compound that comprises a CD43 peptide bed herein, in order to screen for the presence of AML-specific antibodies and/or AML-specific immune cells. If such antibodies or immune cells appear to be t in said sample or in said sample fraction, and to bind said CD43 peptide described herein, said sample is typed as being positive for AML-specific binding compounds (i.e. antibodies and/or immune cells). Said sample for ce comprises a blood sample, or a bone marrow sample, or a biopsy such as for instance a myeloid a (also called a chloroma).

An AML-specific antibody or AML-specific immune cell is for instance detected and/or quantified using an immunoassay, such as for ce a Western blot, a re) ELISA or RIA. These assays are well known in the art. Labelled CD43 peptides bed herein are for instance incubated with a blood sample or bone marrow sample or with or a biopsy such as for instance a myeloid sarcoma, or with a fraction of such sample that comprises antibodies, B cells and/or T cells, where after unbound binding compounds are washed away. Subsequently, it is determined whether the CD43 peptides described herein are bound by AML-specific antibodies or immune cells. In some embodiments, an led CD43 peptide described herein, or an unlabeled compound comprising a CD43 e described herein, is contacted with a sample that comprises antibodies and/or immune cells, such as for instance a blood sample or bone marrow sample or a biopsy such as for instance a myeloid sarcoma, or with a fraction of such sample that comprises antibodies, B cells and/or T cells. After incubation, one or more g steps are preferably performed in order to remove non-bound antibodies and unbound immune cells. Subsequently, it is tested whether antibodies or immune cells have bound said CD43 e described herein, for instance using an dy that is specifically directed against human antibodies or human immune cells and that is coupled to a marker, such as for instance a fluorescent compound or for instance horseradish peroxidase or alkaline phosphatase. After a further washing step, it is determined whether the second antibody has bound, for instance by measuring light emission or by adding a substrate of horseradish peroxidase or alkaline phosphatase.

These detection techniques are well known in the art.

In some ments, a CD43 peptide described herein, or a compound that comprises a CD43 e bed , is contacted with a fraction of a sample that has been enriched for antibodies and/or immune cells. In some embodiments, said on is an in vitro B cell culture or an in vitro T cell culture. In some embodiments, a CD43 peptide described herein or a compound that comprises a CD43 peptide described herein is contacted with antibodies and/or immune cells that have been essentially purified from a biological sample, such as for instance a purified B cell fraction that has been obtained by selecting for CD19 positive cells and/or an antibody/B cell fraction that has been purified using an anti Ig dy or a protein A or G purification method.

Protein A or G purification methods are well known in the art and protocols and ts are commercially ble. As used herein, the term “immune cells that have been essentially purified from a sample” means that at least 80%, preferably at least 85%, more preferably at least 90% or at least 95%, of the cells of a resulting fraction consists of immune cells. The term “antibodies that have been essentially purified from a sample” means that at least 80%, more preferably at least 85%, more preferably at least 90% or at least 95%, of the mass of a resulting fraction consists of antibodies.

Further described is therefore a use of a CD43 peptide bed herein, or a use of a compound that comprises a CD43 peptide described herein, for binding and/or detecting an immune cell and/or an antibody, or a functional part or functional equivalent thereof. Said immune cell and/or antibody or functional part or functional equivalent thereof is preferably able to specifically bind lymphoproliferative cells and/or myeloproliferative cells. Preferably, said myeloproliferative cells are AML cells or MDS cells or CML cells, ably AML cells. A CD43 peptide described herein, or a compound that ses a CD43 peptide described herein, for use as a ion moiety for AML-specific binding compounds such as antibodies and/or immune cells is also herewith described, as well as a method for determining whether a sample comprises AML-specific antibodies and/or AML-specific immune cells, the method comprising incubating a CD43 peptide described herein, or a compound that comprises a CD43 peptide described herein, with said sample, or with a fraction of said sample that comprises antibodies and/or immune cells, and subsequently determining whether said CD43 peptide bed herein is bound by AML-specific antibodies and/or ecific immune cells, or whether said compound that comprises said CD43 peptide described herein is bound by AML-specific antibodies and/or AML-specific immune cells. If such binding is detected, it is concluded that said sample comprises AML-specific antibodies and/or AML-specific immune cells.

Also described is a method for determining whether a sample comprises AML- specific antibodies and/or AML-specific immune cells, the method comprising incubating a CD43 peptide described herein, or a compound that comprises a CD43 peptide described herein, with antibodies and/or immune cells that have been essentially purified from said sample, and subsequently determining r said CD43 peptide described herein is bound by AML-specific antibodies and/or AML-specific immune cells, or whether said compound that comprises said CD43 e described herein is bound by AML-specific dies and/or ecific immune cells.

In some ments, the results of ion tests as described above are used for determining whether an individual has AML. If a sample from an individual s to contain AML-specific immune cells and/or AML-specific antibodies, it can be concluded that said individual is an AML patient. A CD43 e described herein for use as a diagnostic agent is therefore also described herewith, as well as a compound that comprises a CD43 peptide described herein for use as a diagnostic agent. Further described is a use of a CD43 peptide described herein for diagnosing AML, as well as a use of a compound that comprises a CD43 e described herein for diagnosing AML.

Further described is a diagnostic kit comprising: - a CD43 peptide described herein, or a compound that comprises a CD43 peptide described herein, and - means for detecting an antibody-bound CD43 peptide or an immune cell-bound CD43 peptide.

Such means for instance encompass labelled dies that are specifically directed against human antibodies or human immune cells. In some ments, said labelled antibodies are conjugated with horseradish peroxidase or alkaline phosphatase.

Some embodiments describe a diagnostic kit comprising: - a CD43 peptide described herein, or a compound that comprises a CD43 peptide described herein, and - means for detecting an antibody or an immune cell.

Such means for instance encompass ed antibodies that are specifically directed against human antibodies or human immune cells. In some embodiments, said labelled antibodies are conjugated with horseradish dase or alkaline phosphatase.

Some embodiments be a method for typing an antibody-containing sample or an immune cell-containing sample, the method comprising contacting a CD43 peptide described herein (optionally in the context of an MHC x in order to detect T cells), or a compound that comprises said CD43 peptide described herein, with antibodies and/or immune cells of said sample and determining whether said CD43 peptide described herein, or said compound described herein, is bound by at least one of said dies and/or immune cells of said sample. If said CD43 peptide or said compound described herein is bound by antibodies and/or immune cells of said sample, said sample is typed as comprising CD43-specific antibodies and/or immune cells.

Some embodiments be a method for ining whether an individual has a myeloproliferative or lymphoproliferative disorder, preferably AML, the method comprising contacting a CD43 peptide described herein (optionally in the context of an MHC complex in order to detect T , or a compound that comprises said CD43 peptide described herein, with antibodies and/or immune cells of said individual and determining whether said CD43 peptide described herein, or said compound described herein, is bound by at least one of said dies and/or immune cells of said individual.

If said CD43 peptide or said compound described herein is bound by dies and/or immune cells of said dual, it is concluded that said individual has a lymphoproliferative or myeloproliferative disorder such as AML. In some embodiments, said CD43 peptide described herein, or said nd comprising said CD43 peptide described herein, is contacted with a sample that ses antibodies and/or immune cells of said individual, such as for instance a blood sample or a bone marrow sample or a biopsy such as for instance a myeloid sarcoma. In other embodiments, said CD43 peptide or compound described herein is contacted with a fraction of a sample from said individual, wherein said fraction comprises immune cells and/or antibodies. In some embodiments, said CD43 peptide or compound described herein is contacted with antibodies and/or immune cells that have been essentially purified from said sample, such as for instance a purified B cell fraction that has been obtained by selecting for CD19 positive cells and/or an antibody/B cell fraction that has been purified using an anti Ig antibody or a protein A or G purification method.

In some embodiments, the results of detection tests described herein are used for determining whether an individual exhibits a detectable immune response against a myeloproliferative or lymphoproliferative disorder such as AML. This is for instance red for determining whether a patient suffering from a myeloproliferative er who has ed medical treatment, such as for instance an AML patient who has been treated against AML, for instance an AML patient who has received immunotherapy such as a stem cell transplantation or donor-lymphocyte infusion, has a GvL response.

To date, there are no diagnostic tools to test for the presence of a potent GvL response in a treated t. Such diagnostic tool is much , for instance because: 1) It will allow early fication of allogeneic SCT ents at high risk for relapse, at a timepoint before e has occurred y allowing earlier interventions such as tapering of immunosuppressants or donor-lymphocyte infusions; 2) It will allow titrating such donor lymphocyte infusions until anti-leukemia antibodies do appear; and 3) It will offer hope for allogeneic SCT recipients at a time they often suffer from one of many SCT- related complications when the presence of a potent GvL response can be trated.

Nowadays patients have to wait and see whether or not a e , and there is no way to predict relapse of disease. The availability of a test for determining whether a t has an anti-AML immune response will therefore greatly improve the clinical care of SCT patients, affecting prognosis and quality of life.

Some embodiments therefore describe a method for ining whether an individual exhibits an immune response against a myeloproliferative or lymphoproliferative disorder, preferably AML, the method comprising contacting a CD43 peptide described herein, optionally in the context of an MHC x, or a compound that comprises said CD43 peptide described , with antibodies and/or immune cells of said individual and determining whether said CD43 peptide described herein, or said nd comprising said CD43 peptide described herein, is bound by at least one of said antibodies and/or immune cells of said individual. If said CD43 peptide or said compound s to be bound, it indicates that said individual exhibits an immune response against said myeloproliferative or lymphoproliferative disorder, preferably AML. In some embodiments it is determined whether antibodies or B cells of said individual compete with antibody AT14-013 for binding to CD43. Competing dies will be particularly effective against a roliferative or lymphoproliferative disorder, ably AML.

In some embodiments, an antibody or a functional part or a functional equivalent thereof that es with antibody AT14-013 for binding to CD43 is used for detecting myeloproliferative cells in a sample. Further described is ore a use of an isolated, recombinant or purified antibody, or a functional part or a functional equivalent thereof, that competes with antibody AT14-013 for binding to CD43, for determining whether a sample comprises myeloproliferative cells, preferably AML or MDS or CML cells. An isolated, recombinant or purified antibody, or a functional part or a functional equivalent thereof, that competes with antibody AT14-013 for binding to CD43, for use in diagnosis of a lymphoproliferative or myeloproliferative disorder, preferably AML or MDS or CML, is also described herewith, as well as a use of an ed, recombinant or purified antibody, or a functional part or a functional equivalent thereof, that competes with dy AT14-013 for binding to CD43, for diagnosing AML. Also described is a use of an isolated, recombinant or purified antibody, or a functional part or a functional lent thereof, that competes with antibody AT14-013 for binding to CD43, for the preparation of a stic agent for lymphoproliferative or myeloproliferative cells, preferably AML or MDS or CML cells. Some ments describe a diagnostic kit comprising an isolated, recombinant or purified antibody, or a functional part or a functional lent thereof, that competes with antibody AT14-013 for g to CD43, and means for detecting an antibody-cell complex. Said means for instance comprise another antibody against AML cells, such as for instance AT14-013. In some embodiments, said means comprise labelled antibodies against another cell surface component of myeloid cells. In some embodiments, said means comprise labelled antibodies against said antibody or onal part or functional equivalent that competes with antibody AT14-013.

An isolated, recombinant or purified antibody, or a functional part or a functional equivalent f, that competes with antibody AT14-013 for binding to CD43, for use as a diagnostic agent is also described. Some embodiments describe a method for determining whether myeloproliferative cells are present in a sample comprising: - contacting said sample with an isolated, recombinant or purified antibody, or a functional part or a functional equivalent thereof, that competes with dy AT14-013 for binding to CD43, and - allowing said antibody or functional part or functional equivalent to bind myeloproliferative cells, if present, and - determining whether or not myeloproliferative cells are bound to said antibody or functional part or onal equivalent, thereby ining r or not myeloproliferative cells are present in said sample. In some embodiments, said myeloproliferative cells are AML or MDS or CML cells.

Further bed is a use of antibody AT14-013, or a functional part or a functional equivalent f, for determining whether a sample comprises AML or MDS or CML cells. Antibody AT14-013, or a functional part or a functional equivalent f, for use in diagnosis of AML or MDS or CML is also described herewith, as well as a use of antibody AT14-013, or a functional part or a functional equivalent thereof, for diagnosing AML or MDS or CML. Also described is a use of antibody AT14-013, or a functional part or a functional equivalent thereof, for the preparation of a diagnostic agent for AML or MDS or CML.

Other interesting applications of the novel CD43 peptides described herein and nucleic acid molecules or functional equivalents encoding ore are prophylactic or semi-prophylactic applications and immunotherapy. As used herein, a rophylactic application means that an individual already has a disease, but further progression of said disease is at least temporarily delayed or prevented. For instance, a CD43 peptide described herein, or a nucleic acid molecule or functional equivalent thereof encoding therefore, or a vector comprising a nucleic acid molecule or functional lent bed herein, or a compound that comprises a CD43 peptide described herein, can be semi-prophylactically administered to an individual who is suffering from intermediate to high risk ysplastic me (MDS). As described herein before, such patient has an intermediate to high risk to develop AML, so that it is advantageous to elicit an anti-AML immune response in said patient beforehand, using a CD43 peptide or compound or nucleic acid molecule or functional equivalent or vector described herein, before the MDS progresses to AML. Another example of a semi-prophylactic application of a CD43 peptide described herein, or a compound or a nucleic acid molecule or a onal equivalent or a vector described herein, is its use for AML, MDS or CML patients who received an neic poietic stem cell transplantation (HSCT). The goal of such allogeneic HSCT is to evoke an allogeneic graft versus ia/MDS response, but there is currently no approach available to ascertain the development of such se. Also bed is a use of a CD43 peptide described herein, or a nucleic acid molecule or a functional lent ng therefore, or use of a vector comprising a nucleic acid molecule or functional equivalent bed , or use of a compound that comprises a CD43 peptide described herein, as a prophylactic or semi-prophylactic agent that induces an alloreactive immune response against CD43-expressing malignant cells (graft vs tumor response, for example against MDS, AML, or CML). Another example of a prophylactic or semi-prophylactic application of a CD43 peptide bed herein, or a compound or a nucleic acid molecule or a functional equivalent or a vector described , is its use for CML patients. Nowadays, CML is well controlled in many patients using tyrosine kinase tors such as for instance Imatinib. However, a patient may develop resistance to one or more tyrosine kinase inhibitors. Moreover, the use of ne kinase inhibitors sometimes involves adverse side effects like edema, skin rashes, fatigue, nausea and myelosuppression. Tyrosine kinase inhibitors are also expensive. Also described is a use of a CD43 e described herein, or a nucleic acid molecule or a functional equivalent encoding therefore, or use of a vector comprising a nucleic acid molecule or functional lent described herein, or use of a compound that comprises a CD43 peptide described herein, as a prophylactic agent or semiprophylactic 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 described herein is used instead of a tyrosine kinase inhibitor, for instance in order to reduce adverse side-effects and/or costs. In other ments, said CD43 peptide or compound or nucleic acid molecule or functional equivalent or vector described herein is used together with one or more tyrosine kinase inhibitors. A CD43 peptide described herein, or a compound that comprises a CD43 peptide described herein, or a nucleic acid molecule or a functional equivalent f ng a CD43 peptide described herein, or a vector comprising a nucleic acid molecule or functional lent described herein, for use as a prophylactic agent or semi-prophylactic agent is therefore also herewith described. Also described is a use of a CD43 peptide described herein, or use of a compound that comprises a CD43 peptide described herein, or use of a nucleic acid molecule or functional equivalent thereof encoding a CD43 peptide described , or use of a vector comprising a nucleic acid molecule or functional equivalent described herein, for the preparation of a prophylactic agent or semi-prophylactic agent against AML, for instance for an AML patient that has received allogenic HSCT. In some embodiments, said semi-prophylactic agent is for a MDS or CML patient, as explained above. Said prophylactic agent or semi-prophylactic agent preferably comprises a vaccine. As used , the term “prophylactic agent” also encompasses semiprophylactic agents.

In some embodiments, a CD43 peptide or compound or nucleic acid molecule or functional equivalent or vector bed herein is used for treatment of a myeloproliferative or lymphoproliferative disorder, preferably AML. As used herein, “treatment” encompasses ation of at least one symptom, and/or delaying or even halting the progression of disease, at least temporarily. In one preferred embodiment, a CD43 peptide described herein, optionally in the context of an MHC complex, or a nucleic acid molecule or a functional equivalent encoding therefore, or a vector sing a nucleic acid molecule or functional equivalent bed herein, or a compound that comprises said CD43 peptide described herein, is administered to an AML patient in order to boost his/her immune system, resulting in an enhanced immune response. In some embodiments, naïve T cells or B cells from an AML patient are cultured ex vivo and incubated with a CD43 peptide or nd described , optionally in the context of an MHC complex in case of a T cell culture, in order to obtain AML-specific T cells or B cells that are subsequently administered to the patient, optionally after ex vivo ion. In some embodiments it is determined whether AML- specific B cells from said AML patient compete with antibody 13 for binding to CD43, because competing B cells will be particularly effective against AML, in particular in view of the fact that AT14-013 also targets ic stem cells, which are known to be more y resistant and often responsible for relapse of disease after ent.

In some embodiments, adoptive cell therapy is used. In some embodiments, T cells from an AML patient are tested for binding or activation using a CD43 e described herein in the context of an MHC complex, or using a compound comprising a CD43 peptide described herein in the t of an MHC complex, and T cells izing said CD43 peptide are preferably expanded ex vivo and subsequently administered to the patient, which will result in an anti-AML T cell response.

In some ments, adoptive cell therapy of donor lymphocytes is used. Donor T cells isolated from an AML patient who received allogeneic HSCT, or isolated from the HSCT donor, are preferably tested for binding or activation using a CD43 peptide described herein in the context of an MHC complex, or using a compound comprising a CD43 peptide described herein in the context of an MHC complex, and donor T cells recognizing said CD43 peptide are preferably expanded ex vivo and subsequently administered to the patient, which will result in an anti-AML allogeneic T cell response.

In some embodiments, T cells are modified in order to be them with an AML-specific binding moiety. Said T cells are preferably derived 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 produced. These are T cells with modified T cell receptors, which have been provided with a binding specificity of interest, preferably derived from an antibody. Typically, CAR T cells are produced by fusing a single-chain variable domains (scFv) derived from a monoclonal antibody to the CD3-zeta transmembrane domain, so that a zeta signal will be elicited upon target ition by the scFv. ing to some embodiments, a CD43 peptide described , or a nucleic acid le or a functional equivalent encoding therefore, or a vector comprising a nucleic acid molecule or functional equivalent described herein, or a compound that comprises a CD43 peptide described , is used in order to produce and/or isolate a CD43-specific B cell and/or antibody, which in turn is used for the production of a modified T cell. For instance, said CD43 peptide or compound or nucleic acid molecule or functional equivalent or vector is used in order to elicit, detect and/or isolate an AML- specific antibody or AML-specific B cell. Subsequently, in some embodiments the heavy chain and/or light chain variable domains of said dy or B cell are provided to T cells, thereby producing modified T cells with an AML specificity. In some embodiments, these ed T cells are subsequently stered to an AML patient, which will result in an AML-specific T cell response. In some embodiments, said modified T cells are CAR T cells. In some embodiments said AML-specific antibodies or AML-specific B cells are tested for competition with dy 13 for binding to CD43 before the heavy chain and/or light chain variable domains of said antibodies or B cells are ed to T cells. Such competing antibodies are preferably selected for ing modified T cells with an AML specificity.

Further described is therefore a CD43 peptide described herein, optionally in the context of an MHC complex, or a nd that comprises said CD43 peptide described , or a nucleic acid molecule or functional lent f encoding a CD43 peptide described herein, or a vector comprising a nucleic acid molecule or functional equivalent described herein, for use as a medicament. Also described is a use of a CD43 peptide described , optionally in the context of an MHC complex, or a compound that comprises said CD43 peptide described herein, or a nucleic acid le or functional equivalent thereof encoding a CD43 peptide described herein, or a vector comprising a nucleic acid molecule or functional lent described herein, for the production of AML-specific T cells. Some embodiments describe a method for producing a ed T cell, the method comprising ting an antibody-containing sample from an AML patient or a B cell-containing sample from an AML t with a CD43 peptide or compound described herein, resulting in bound antibodies or B cells against AML, and subsequently ing one or more AML-specific domains from an ecific antibody or from an AML-specific B cell from said AML patient and providing said one or more domains to a T cell. Some embodiments describe a method for producing a modified T cell, the method comprising immunizing a non-human animal with a CD43 peptide or compound or nucleic acid molecule or functional equivalent or vector described herein, thereby eliciting an immune response against AML, and subsequently obtaining one or more AML-specific domains from an AML-specific antibody or an AML-specific B cell from said man animal, or obtaining one or more nucleic acid sequences encoding for said one or more AML-specific domains, optionally after it has been determined whether said AML-specific antibody or AML-specific B cell competes with antibody AT14-013 for binding to CD43, and providing said one or more domains, or said one or more nucleic acid sequences, to a T cell. A CD43 peptide or compound bed herein for use in immunotherapy is also described herewith, as well as a CD43 peptide described herein in the context of an MHC complex for use in immunotherapy, as well as a nucleic acid molecule or onal equivalent thereof encoding a CD43 peptide described herein for use in immunotherapy. Some embodiments describe a vector comprising a nucleic acid molecule or functional equivalent described herein for use in immunotherapy. Further described is a use of a CD43 e bed herein, or a use of a compound that comprises a CD43 peptide described herein, or a use of a nucleic acid molecule or functional equivalent thereof encoding a CD43 peptide described herein, or a use of a vector comprising a nucleic acid molecule or functional equivalent described herein, for the preparation of a medicament against a myeloproliferative or lymphoproliferative disorder, preferably AML.

Also bed is an genic composition sing a CD43 peptide described herein, and/or comprising a compound that comprises a CD43 peptide described herein, and/or sing a nucleic acid molecule or functional equivalent thereof encoding a CD43 e described herein, and/or comprising a vector that comprises a nucleic acid molecule or functional equivalent described herein. Said immunogenic composition preferably further comprises a patible additive, such as for instance a r, diluent, excipient or filler. Some embodiments describe a vaccine comprising a CD43 peptide described herein, ally in the context of an MHC complex. Some embodiments describe a vaccine comprising a compound that comprises said CD43 peptide described herein, and a vaccine sing a nucleic acid molecule or functional equivalent thereof encoding a CD43 peptide described herein, and a vaccine comprising a vector that comprises a nucleic acid molecule or functional lent described herein. Other embodiments describe a composition comprising a CD43 peptide bed herein, or a composition comprising a nd that comprises a CD43 peptide described herein, or a composition comprising a nucleic acid molecule or onal lent thereof encoding a CD43 peptide described herein, or a composition comprising a vector that ses a nucleic acid molecule or functional equivalent described herein, n said composition is a pharmaceutical composition which further comprises a pharmaceutically acceptable carrier, diluent or ent.

In some embodiments, an isolated, recombinant or purified antibody, or a functional part or a functional equivalent thereof, that competes with antibody AT14-013 for binding to CD43 is used for treatment of a myeloproliferative or proliferative disorder, preferably AML. As described in , antibody AT14-013 was obtained from an AML patient in complete remission, demonstrating that AT14-013 is effective against AML. Moreover, AT14-013 also targets leukemic stem cells, which are known to be more therapy resistant and often responsible for relapse of disease after treatment. Antibodies that compete with AT14-013 for g to CD43 will therefore also be very effective against myeloproliferative disorders like AML. Hence, stration of such antibodies to an AML patient will effectively counteract, and/or kill, AML cells. Some embodiments therefore describe an isolated, recombinant or purified dy, or a onal part or a functional equivalent thereof, that competes with antibody AT14-013 for binding to CD43, for use as a medicament. Some embodiments describe a use of an isolated, recombinant or purified antibody, or a functional part or a functional equivalent thereof, that competes with antibody AT14-013 for binding to CD43, for the preparation of a medicament.

Also described is an isolated, recombinant or purified antibody, or a functional part or a functional equivalent f, that competes with antibody AT14-013 for binding to CD43, for use in a method for at least in part treating or preventing a myelodysplastic or myeloproliferative or lymphoproliferative disorder, as well as a use of an isolated, inant or purified antibody, or a functional part or a functional equivalent thereof, that competes with antibody AT14-013 for binding to CD43, for the preparation of a medicament against a roliferative or proliferative disorder. Said roliferative disorder preferably comprises AML. Further ments describe a composition comprising an isolated, recombinant or purified antibody, or a functional part or a functional equivalent f, that competes with antibody AT14-013 for binding to CD43. Said composition is preferably a pharmaceutical composition that comprises a pharmaceutically acceptable carrier, diluent or excipient As described herein before, some embodiments describe an isolated, synthetic or recombinant nucleic acid molecule, or a functional equivalent thereof, encoding a CD43 peptide described herein. Such nucleic acid molecule or functional equivalent is for instance useful for the tion of a CD43 peptide described herein, using a nucleic acid expression system such as for instance 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, cells selected from the group consisting of Kasumi 3 cells, HL60 cells, KG1a cells, SH2 cells, c6 cells, Molm13 cells, and CML K562 cells are used as host cells.

Said nucleic acid le or functional equivalent described herein is also useful for eliciting an immune response. For instance, a nucleic acid molecule or functional equivalent described herein is stered 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 described herein is administered to an MDS or CML patient in order to delay or prevent the progression of MDS or CML to AML (prophylactic or semi-prophylactic applications). In some embodiments, a nucleic acid molecule or onal equivalent described herein is administered to a non-human animal in order to elicit an anti-AML immune response, where after AML-specific antibodies and/or AML-specific immune cells can be harvested from said animal.

Alternatively, the variable domain-encoding nucleic acid sequences of AML-specific B cells from said non-human animal are sequenced in order to obtain one or more nucleic acid sequences of AML-specific variable domains, where after one or more nucleic acid molecules sing AML-specific variable domain sequences are introduced in producer cells for the production of AML-specific antibodies.

In some embodiments, a nucleic acid molecule or functional equivalent described herein is present in a gene delivery vehicle, which facilitates introduction of said nucleic acid molecule or functional equivalent into a cell of interest. Further described is ore a gene delivery vehicle, preferably a vector, comprising a nucleic acid molecule or functional equivalent described herein. A host cell comprising a nucleic acid molecule or functional equivalent described herein, and/or comprising a gene delivery vehicle or vector described , is also described herewith.

While the t application may describe features as part of the same embodiment or as parts of separate embodiments, the scope of the present invention also includes embodiments sing any combination of all or some of the es described herein.

The term “comprising” as used in this specification and claims means “consisting at least in part of”. When interpreting statements in this specification, and claims which include the term “comprising”, it is to be understood that other es that are additional to the features ed by this term in each statement or claim may also be present. Related terms such as “comprise” and “comprised” are to be interpreted in similar manner.

In this specification where reference has been made to patent specifications, other al documents, or other sources of information, this is generally for the purpose of ing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be ued as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

In the description in this specification reference may be made to t matter that is not within the scope of the claims of the current application. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the claims of this application.

The invention is further explained in the following Examples. These Examples do not limit the scope of the ion, but merely serve to clarify the invention.

Brief description of the drawings Figure 1. ce of AT14-013 (2K23-1K13) including the variable heavy and light chain sequences and the CDR sequences of the antibody.

Figure 2. Binding of AT14-013 to AML cell lines and freshly isolated primary AML blasts from newly sed patients. FAB: -American-British classification of AML tt et al. 1976).

Figure 3. AT14-013 binds to AML cell lines and primary isolated AML cells.

Representative examples of binding of AT14-013 derived from patient 101 to the AML cell lines Kasumi3, SH-2, Molm13 and THP-1 and to primary leukemic blasts isolated from newly diagnosed AML patients (FAB classification M0-M5). An in-house produced human antibody specific for influenza was used as a negative control (grey filled histograms).

Figure 4. AT14-013 also binds to leukemic blasts from patients with isk myelodysplastic syndrome (MDS/RAEB I and II) and blast crisis chronic myeloid leukemia (CML). Depicted are representative examples; indicated are patient identification codes except K562 which is a CML cell line. An in-house produced human antibody against influenza was used as a negative l (grey filled histograms).

* BL-060: biphenotypic leukemia, responding well to AML treatment.

Figure 5. AT14-013 does not bind to eloid cells. (a) AT14-013 did not bind to healthy PBMCs, T cells , B cells (CD19+), non activated monocytes ) or primary isolated thymocytes (except for a small population of d cells that are present in fetal thymus). (b) AT14-013 also did not bind primary isolated B- or T- ALL cells, lymphoma’s or multiple myeloma. (c) AT14-013 also did not bind colon carcinoma cell lines or primary isolated cells from patients with colon carcinoma (Colon CA) or healthy colon or ileum.

AT14-013 did bind to granulocytes (a) and human melanoma cell lines (c). An se produced human antibody against influenza was used as a negative control (grey filled histograms).

Figure 6. CDC and ADCC. Calcein labeled THP-1 cells were incubated with AT14-013 and rabbit serum complement. Living cells were identified as calcein+, dapicells.

With our bead based assay the amount of dead cells could then 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 is also able to induce antibody ent cell cytotoxicity (ADCC) in a Jurkat reporter system with the AML cell line SH-2 or freshly isolated leukemic blasts as target cells (right panel).

Figure 7. Target identification of AT14-013: precipitation (IP). IP with biotin-labeled (via a sortase tag) AT14-013 of THP1 cell lysates yielded a ~140kDa band on an Imperial Coomassie stained gel. The band is specific as it is not seen in the AT10- 002 IP of THP1 lysate or in the Jurkat lysate IP. The band was excised from gel and the target ified as CD43 by mass spectrometry.

Figure 8. Target mation of AT14-013. THP-1 and Molm13 lysates were immunoprecipitated with AT14-013 or with the influenza-specific antibody AT10-002.

Western blot analysis with mouse-anti-CD43 (clone Mem59) confirmed CD43 as the binding target of AT14-013.

Figure 9. AT14-013 binds to a unique CD43 epitope. (a) THP-1 cells were stained with the 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 dies. In Kim ea, (Kim et al. 2014), binding of cially ble CD43 antibodies YG5, 2C8, 8E10 and DFT-1 to various cell lines is summarized. We compared binding of AT14-013 to the same cell lines and found a different binding pattern. (c) A competition experiment with AT14-013 and commercially available CD43 specific antibodies was performed as indicated. Briefly, THP-1 cells were incubated with indicated antibodies at increasing concentrations, after which the possibly competing antibody (referred to as ‘competing dy’) was added. AT14-013 binding to THP-1 target cells was not affected by pre-incubation of the cells with commercially available CD43 antibodies, while these commercially ble CD43 dies did inhibit each other’s g to THP-1 cells. Results are shown for experiments wherein AT14-013 or 84-3C1 was the “competing antibody”. (d) Summary of competition experiments.

AT14-013 does not compete with commercially available CD43 antibodies for binding to THP-1, ting that AT14-013 binds a different epitope.

Figure 10. osylation of THP-1 cells with neuraminidase (sialidase) s the sialic acids from the cell membrane. “No” indicates no inidase treatment and “1:20” and “1:200” indicates the neuraminidase dilution. Antibodies AT14-013, Mem59, DF-T1 and 84-3C1 lost binding to THP-1 cells after neuraminidase treatment of these cells. Clone L10 is not binding to a sialilated epitope of CD43, as neuraminidase treatment of THP-1 cells did not affect g of this antibody to its target cells.

Figure 11. CD43 truncated variants map the epitopes of commercially available antibodies DF-T1 and MEM59. a) blot of HEK293T cells expressing truncated variants of CD43 probed with anti-CD43 directed towards the intracellular C-terminal tail of the protein. b) Immunostaining of the same blot with CD43 specific antibodies MEM59 (upper panel) and DF-T1 (lower panel) revealed the presence of their e in region ‘C’ (amino acids .

Figure 12. Immunoprecipitation of CD43 truncated variants from THP1 cells identifies the AT14-013 epitope. a) Immunoblot of input lysates of sorted CD43 truncated variant overexpressing THP1 cells probed with anti-Flag antibody. uS staining demonstrates equal loading of s. All mutant proteins are sed. b) Anti-Flag blot of eluted immunoprecipitations of THP1 variant cell lines with AT14-013 reveals binding to mutants A-F and no binding to mutants H-J, defining the epitope. c) Immunoblot with anti-CD43 cytoplasmic tail binding antibody ) showing endogenous immunoprecipitated CD43 in all samples as well as staining of truncated variants.

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

Figure 14. Binding of AT14-013 to other AML blasts. Binding of AT14-013 to freshly isolated primary AML blasts (CD45dim) from newly diagnosed patients. An in-house produced human antibody specific for influenza was used as a negative control. For the commercial mouse anti CD43 antibodies a mouse anti CMV was used as control.

WHO: Swerdlow S.H. WHO classification of Tumours of Haematopoietic and Lymphoid tissues (2008). CD43+ T cells and tonsil cells were used as extra control for the assay.

AT14-013 does not bind to these healthy cells. (% gated = -;<10%, +; 10∼25%, ++; 25∼50%, +++; 50∼75%, ++++; 75∼100%) Figure 15. ADCC and CDC. a) AT14-013 (open s) is e of inducing antibody dependent cell ed xicity (ADCC) on the AML cell line SH-2 with PBMCs in an effector target ratio of 50:1. Living cells were identified as calcein+, dapi- cells. With our bead based assay the amount of dead cells could then be calculated. Incubation of SH2 cells with AT10-002 did not induce ADCC (black dots). The calculated EC50 for AT14-013 is 0,16ug/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 fied as calcein+, dapi- cells. Incubation of SH2 cells or AML blasts with AT10-002 did not induce CDC (black dots). The calculated EC50 for AT14-013 was 1,86 ug/ml.

Figure 16. a) Anti-Flag immunoblot of eluted immunoprecipitations of THP1 t cell lines with AT14-013 reveals binding to mutants A-F2, binding to a lesser extent to G, and no binding to mutants H-J. b) Immunoblot with anti-CD43 cytoplasmic tail binding antibody (Novus) showing nous immunoprecipitated CD43 in all samples as well as staining of truncated variants. This control confirms that the immunoprecipitation was successful for all samples shown.

Figure 17. a) Treatment of mice engrafted with SH-2 AML cells leads to a tumor growth inhibition of 90.3 % as measured at the ice by whole body measurement 01, repeated ANOVA). b) The number of AML cells, measured by the number of photon per minute (cpm) exhibits a strong decrease in all the organs measured (p=0.0011, repeated 2way ANOVA). c) Evaluation of the number of tumor cells by FACS in the bone marrow and the liver (p=0.0017, 2way ANOVA).

Figure 18.

Representative examples of g of AT14-013 to fetal CD34+ hematopoietic stem cells (HSC) but not to fetal CD34+CD38+ progenitor cells or fetal CD34-CD38- mature cells.

Grey filled histograms: control antibody AT10-002 directed against influenza, described in .

Figure 19.

AT14-013 reacts with gous leukemic stem cells.

AML blasts of donor # 101 (the same donor from whom the B cells ing AT14-013 were obtained) were stained with AT14-013 and with antibodies specific for CD34 and CD38, and with an antibody against CD45 (BD, cat 348815) to distinguish the general blast population (CD45 dim) from healthy cells in the bone marrow and analyzed by flow cytometry.

Examples MATERIAL & METHODS Patient and healthy human als Study protocols were approved by the Medical Ethical Committee of the Academic Medical Centre. All participants signed informed consent. Participants included healthy individuals and patients with hematologic malignancies recruited from our clinic that donated peripheral blood and/or bone marrow.

Generation of AML-specific clone AT14-013 As described in Example 2 of , transduced naïve and memory IgG B cells of AML patient 101, immortalized by introduction of Bcl6 and Bcl-xL as described usly enbos et al., Nat Med 2010 and Example 1 of ), were seeded at a concentration of 20 or 40 cells per well (hereafter named microcultures) and expanded with IL-21 and CD40L. Supernatants of expanded B cell microcultures were then screened for dy binding to AML cell lines (amongst others THP-1, MonoMac6), and to liver and colon cell lines, by FACS, using human IgG H+L AF647 (Life Technologies) or human-IgG-PE ern Biotech) as a secondary antibody.

Several in-house generated antibodies were used as negative control antibodies, such as anti-CD30 (expressed on activated B and T lymphocytes), D33 ssed on monocytes, myeloid progenitor cells and d leukaemias), D25 (against RSV; described in ) and AT10-002 (against nza; described in ). Microcultures binding to AML cell lines but not to liver and colon cell lines were selected and seeded at a tration of 1 cell/well and their supernatants tested again for specificity for AML cell lines. Clones with supernatants specifically binding AML cell lines and not liver or colon cell lines, or healthy PBMC and bone marrow were selected for sequencing. Clones were expanded under normal culture conditions in the presence of FBS IgG low serum (Hyclone) and antibodies purified from the supernatants of these cultures as described below for the recombinant antibodies.

The recombinant antibodies were then again tested for specific binding. One of the obtained AML-specific antibodies was AT14-013. The ered AT14-013 antibody was additionally tested on many freshly isolated blasts of newly diagnosed AML patients (FAB M0-M5) for binding, using human IgG H+L AF647 (Life Technologies) as a secondary antibody.

Cloning of AML-specific antibody AT14-013 As described in Example 1 of , to produce recombinant antibody we isolated total RNA with the ® mini kit (Qiagen), generated cDNA, performed PCR and cloned the heavy and light chain variable regions into the pCR2.1 TA cloning vector (Invitrogen). To rule out reverse transcriptase or DNA polymerase induced mutations, we med several independent cloning experiments. To produce recombinant mAb we cloned heavy and light variable regions of each antibody in frame with human IgG1 and Kappa constant regions into a pcDNA3.1 (Invitrogen) based vector and transiently transfected 293T cells. We purified recombinant antibodies from the culture supernatant with Protein A or G, depending on the Ig subtype of the clone.

CDC and ADCC To quantify complement dependent cell death (CDC) of target cells induced by AML- specific antibody AT14-013 we used a FACS-based leukemia cell lysis assay. THP-1 cells were incubated with 2 μM Calcein AM (Becton Dickinson) for 30 minutes at 37°C.

Calcein labeled THP-1 cells were incubated together with antibodies and rabbit serum complement for 4 hours at 37°C. FACS calibration beads (Accudrop Fluorescent Beads, BD Biosciences) were added to the cells in a 50/50 ratio after which a rd amount of beads was acquired with FACS. As an equal assay volume was ained by the calibration beads, the amount of dead cells was calculated as: 100 - ((Dapi negative, Calcein AM ve cells in respective treatment/Dapi negative, Calcein AM positive cells in control) × 100). For the dy dependent cell mediated cytotoxicity (ADCC) we generated a read-out system with Jurkat cells that were stably transduced with NFAT(6x)-IL2 (minimal promoter)-GFP and CD16a IIa). Activation of the CD16a receptor by bound antibody in this system activates NFAT which induces GFP expression that is then used as a read-out to fy effector cell activation. AML cells t cells) were ted with dies and mixed with Jurkat cells (effector cells) that were stained with Calcein AM as described above. Effector : target ratio was 1:1.

AT14-013 target fication and validation THP-1 cells were lysed (0,5% Triton X114 (Sigma), 150mM NaCl, 10mM Tris-HCL pH7.4, 1,5mM MgCl2 supplemented with protease and phosphatase inhibitors (Roche)) and ared with an irrelevant antibody (in-house generated RSV antibody D25), Protein-G and Streptavidin beads (Pierce) to remove non specific binding proteins.

Precleared lysate was then incubated with bead-bound AML-specific antibodies or with the influenza specific antibody AT10-002 as a negative control (3 hrs at 4°C). Antibody- incubated beads were washed five times in lysis buffer supplemented with 0,5% Deoxycholate and 0,1% SDS, bound proteins were eluted from the beads (0,1M Glycine pH10,5, 150mM NaCl, 1% Triton X100, 1mM EDTA) and then run on an GE gel. 85% of IP samples was run on SDS-PAGE and stained with Imperial protein stain (Pierce) to stain total proteins and excise specific bands for Mass Spectrometry. The rest of the IP samples were run on SDS-PAGE and transferred to PVDF membrane (Bio- RAD) for immunoblotting. The blot was stained with Ponseau S to reveal total protein and blocked with BSA, then incubated with mouse-anti-CD43 (clone MEM-59, Abcam) for Western blot analysis.

Epitope mapping: competition THP-1 cells were pre-incubated for 60 minutes on ice with AT14-013 and the commercially available CD43 antibodies: mouse anti human CD43 PE (Ebioscience; clone 84-3C1), mouse anti human CD43 FITC (Invitrogen; clone L10), mouse anti human CD43 FITC (Abcam; clone ), mouse anti human CD43 unlabeled ; clone MEM-59), mouse anti human CD43 unlabeled (Thermo Scientific; clone DF-T1). The maximum blocking antibody concentration was 10ug/ml. Next, the ing antibody was added with a final concentration of 1 ug/ml. With this step, the final concentration of the blocking antibodies is 2ug/ml. Cells were incubated for 30 minutes on ice, after which dapi (Sigma) was added to exclude dead cells from the analyses. Samples were analyzed by flow cytometry.

Epitope mapping: deglycosylation THP-1 cells were incubated with neuraminidase ; dilution 1:20 or 1:200) for 60 minutes at 37°C to remove sialic acids from CD43 (de Laurentiis et al. 2011). Cells were then washed, blocked in 60% normal goat serum and ted with AT14-013 and the commercially available CD43 antibodies DF-T1, , L10 and MEM-59 as described above. To allow ison of cell staining with different fluorochromes, binding to untreated cells (no inidase) was set to 1. Depicted in Figure 10 is fold increase / se of g to neuraminidase treated cells.

Epitope mapping: CD43 truncated variants.

CD43 cDNA was obtained from Geneart (Life Technologies) and adapted to contain a 3xFLAG tag in-frame on either C- or N-terminus (C-terminal to the signal peptide, sing the first 19 amino acids of CD43). The cDNA was cloned into the pHEF-TIG third-generation lentiviral vector ning an IRES-GFP 3’ of the 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 for GFP to obtain a pure population of CD43 overexpressing cells. Truncated CD43 variants were constructed by PCR-cloning of the CD43 C-terminal FLAG-tagged cDNA to contain the signal peptide (AA 1-19) followed by the wild-type full length extracellular sequence (variant A: S20-P400, followed by 3xFLAG: DYKDHDGDYKDHDIDYKDDDDK) or truncated extracellular sequences (variant B-J). B: 31-400; C: 59-400; D: 82-400; E: 112- 400; F: 133-400; G: 166-400; H: 184-400; I: 202-400; J: 220-400 (the embrane domain starts at AA 255. These variants were expressed in THP1 cells by lentiviral transduction and GFP . Sorted cells were lysed and immunoprecipitated with AT14-013 and l as described above. Eluted IP samples were run on SDS-PAGE and immunoblotted with anti-FLAG-HRP (Sigma) to reveal binding.

RESULTS 13 specifically binds to AML cells In this Example we identify the target of the AML specific antibody 13 that was recently developed in our laboratory ( and figure 1). This antibody is derived from a patient called t 101. He was sed with an ediate-risk AML (no cytogenetic or molecular abnormalities; FAB classification AML-M5) at the age of 49 years. He received two courses of chemotherapy (cytarabine, idarubicine, amsacrine) and one course of idation chemotherapy (busulphan, cyclophosphamide) followed by an autologous hematopoietic stem cell lantation (HSCT), as there was no HLA-matched sibling stem cell donor ble. Fourteen months after the first diagnosis his disease relapsed. He obtained complete remission after one cycle of high-dose cytarabine, after which he received a reduced intensity allogeneic HSCT of a matched, unrelated donor (RIST-MUD). Six weeks later he developed acute GvHD of skin, liver and intestine (stage 1; grade II) that responded well to corticosteroid therapy. Given the fact that this patient remained disease free for over years now, despite the high-risk nature of his disease, this patient can be considered to have generated a potent graft versus AML response which was the reason he was selected to search for potent AML-specific antibody responses. B cells were ed from a phlebotomy product obtained from this patient 38 months post-HSCT, immortalized by introduction of Bcl6 and Bcl-xL as described previously (Kwakkenbos et al., Nat Med 2010) and cultured in 20 or 40 cells/well concentrations. Supernatants of these microcultures were screened for binding to AML cell lines and microcultures specific for AML subcloned in one cell/well concentrations. One of the dies identified through this procedure is 13, an IgG1 kappa, highly somatic hypermutated antibody.

AT14-013 binds specifically to a wide variety of AML cell lines and primary AML cells, covering all AML FAB classifications, as shown in figure 2. In figure 3, a number of representative es of AT14-013 binding to Kasumi3, SH-2, Molm13 and THP-1 and to y leukemic blasts isolated from newly diagnosed AML patients are shown.

In addition, AT14-013 binds to other myeloid malignancies such as AML from high-risk myelodysplastic syndrome (MDS/RAEB I/II) or blast crisis chronic myeloid leukemia (CML) and the CML cell line K562 e 4). AT14-013 did show some binding to granulocytes but did not bind to healthy peripheral blood mononuclear cells (PBMC), bone marrow, thymocytes, hematologic malignancies of the lymphatic e or healthy or malignant cells of liver and colon. AT14-013 did bind to cultured melanocytes and melanoma cell lines (figure 5).

AT14-013 induces CDC and ADCC of target cells AT14-013 can induce complement ent cytotoxicity and antibody dependent cellular cytotoxicity (figure 6) of AML cell lines and primary isolated AML blasts.

The target of AT14-013 is a unique epitope of CD43 We then identified the target of AT14-013. precipitation (IP) of THP-1 lysate incubated with biotin-labeled sortase-tagged AT14-013 yielded a ~140kDa band. The band is ic as it was not seen in the AT10-002 IP of THP1 lysate nor in the Jurkat lysate IP (figure 7). Mass-spectometry analysis of the immunoprecipitation band ed CD43 as the target protein. Three out of three expected intracellular peptides were identified, giving a 7% coverage of the protein, extracellular peptides were not identified since these are heavily glycosylated. CD43 binding by AT14-013 was med by western blot is. Briefly, THP-1 and Molm13 lysates were immunoprecipitated with 13 or with the influenza-specific antibody AT10-002.

Western blot analysis with mouse-anti-CD43 (clone Mem59) confirmed CD43 as the binding target of AT14-013 (figure 8).

CD43 is widely expressed on healthy and malignant cells. CD43-specific antibodies have been generated and are commercially available, such as DF-T1, 84-3C1, L10 and MEM- 59. With these antibodies we confirmed CD43 expression by THP-1 cells (figure 9a). The observation that AT14-013 does not bind to non-myeloid cells and the different binding profile of AT14-013 to all sorts of cells and cell lines compared to other CD43 antibodies (figure 9b) suggests that AT14-013 recognizes a different CD43 epitope than the other CD43 antibodies. Indeed, when we performed competition experiments, incubating THP- 1 cells with commercially available CD43 dies and AT14-013, we found that these CD43 antibodies compete with each other for binding to THP-1, but not with AT14-013 (figure 9c and figure 9d). Of note, CD43 clones L10 and 84-3C1 have been described to compete with each other (L. Borche et al 2005); this is confirmed in our ment.

The CD43 protein is a highly glycosylated protein (de Laurentiis et al. 2011). The CD43 antibodies Mem59, DF-T1 and 84-3C1 (but not L10) bind to a sialylated epitope, as after pretreatment of target cells with neuraminidase, which removes all α-N-acetylneuramic acids c acids), binding of these dies to CD43 is lost (US2010/0234562A1). In figure 10 we trate that binding of 13 to THP-1 cells is also lost upon preincubation of THP-1 cells with neuraminidase, demonstrating that 13 specifically binds to a sialylated epitope of CD43.

To more specifically identify the binding epitope of AT14-013, we generated 10 Flagtagged extracellular-truncated variants of CD43 that were expressed in HEK and THP1 cells. Western blot analysis of lysates of these cells incubated with Mem59 or DF-T1 confirmed g of these antibodies to a similar epitope between amino acids 59-82 e 11a,b). We tested 13 binding by immunoprecipitation of THP1 cells transduced with these truncation variants. AT14-013 interacts strongly with ts AF , to a lesser extent with variant G, and not with variants H-J as shown in the anti-Flag immunoblot of the IP’s (Figure 12a,b). In figure 12c we med the AT14-013 IP with an anti C terminal CD43 antibody. In all samples endogenous CD43 was present, whereas there was only truncated CD43 present up to variant G. We therefore conclude that the epitope of AT14-013 lies between amino acids 133 and 184. e 2; Binding to AML blasts Material & methods Binding of antibody AT14-013 to different cells was tested using the methodology as described in Example 1 under the heading ‘Generation of AML-specific clone AT14-013’.

Patient samples were stained with anti human CD45 (BD) prior to the assay. AML cells were defined as CD45dim. Healthy PBMCs were stained with anti human CD3 (biolegend). Polymorph nuclear cells d from tonsil were isolated by ficol density gradient.

RESULTS AT14-013 binds specifically to a wide variety of AML cell lines and primary AML cells, covering all AML FAB classifications, as shown in Example 1 and Figure 4. Additionally, we tested the antibody on a broader panel of AML blasts. It showed to bind to all AML blasts tested so far and often better than the commercial anti CD43 antibodies did.

Interestingly, the sialic acid ndent L10 antibody was binding the least in almost all samples. In on the antibodies were tested on healthy CD43 expressing T cells and cells derived from tonsil. Here, only the commercial antibodies showed staining. The results are summarized in Figure 14.

Example 3; ADCC and CDC In addition to Example 1 and Figure 6, r ADCC and CDC experiment was performed.

Material & methods To fy antibody dependent cell-mediated xicity (ADCC) and complement dependent cellular cytotoxiciy (CDC) of target cells induced by AML-specific antibody AT14-013 we used a FACS-based leukemia cell lysis assay. SH2 cells were incubated with 10 nM Calcein AM (Becton Dickinson) for 30 minutes at 37°C. Calcein d cells were then incubated together with antibodies and y peripheral blood mononuclear cells (PBMCs; or:Target 50:1) for 4 hours or rabbit serum complement for 1 hour at 37°C. FACS calibration beads (Accudrop Fluorescent Beads, BD Biosciences) were added to the cells in a 50/50 ratio after which a standard amount of beads was acquired with FACS. As an equal assay volume was ascertained by the ation beads, the amount of dead cells was calculated as: 100 - ((Dapi negative, Calcein AM positive cells in respective treatment/Dapi negative, Calcein AM positive cells in l) × 100).

S AT14-013 induces CDC and ADCC of target cells 13 can induce antibody dependent cell mediated cytotoxicity (figure 15A) and induce complement dependent cytotoxicity (figure 15B) of AML cell lines and primary isolated AML blasts.

Example 4; Epitope g: CD43 truncated variants In addition to e 1 and Figure 12, the binding epitope of AT14-013 was further investigated.

Material & methods The same methods as in Example 1 were used. CD43 cDNA was obtained from Geneart (Life Technologies) and adapted to contain a 3xFLAG tag in-frame on either C- or N- terminus (C-terminal to the signal peptide, comprising the first 19 amino acids of CD43).

The cDNA was cloned into the pHEF-TIG third-generation lentiviral vector containing an FP 3’ of the 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 for GFP to obtain a pure population of CD43 transduced cells. Truncated CD43 variants were constructed by PCR-cloning of the CD43 C-terminal FLAG-tagged cDNA to contain the signal e (AA 1-19) followed by the wild-type full length extracellular sequence (variant A: S20-P400, followed by 3xFLAG: DYKDHDGDYKDHDIDYKDDDDK) or truncated extracellular sequences (variant B-J).

B: 31-400; C: ; D: 82-400; E: 112-400; F: 133-400; F2: 148-400; G: 0; H: 184- 400; I: 202-400; J: 220-400 (the transmembrane domain starts at AA 255). These variants were expressed in THP1 cells by lentiviral transduction and GFP . Sorted cells were lysed and immunoprecipitated with AT14-013 and control as described above.

Eluted IP samples were run on SDS-PAGE and immunoblotted with anti-FLAG-HRP (Sigma) to reveal binding.

RESULTS The target of AT14-013 is a unique epitope of CD43 To more specifically identify the binding e of 13, we generated 11 Flagtagged extracellular-truncated variants of CD43 that were expressed in THP1 cells. We tested 13 binding by immunoprecipitation of THP1 cells transduced with these truncation variants. AT14-013 interacts strongly with variants A-F, to a lesser extent with variant F2, to a lesser extent with variant G, and not with variants H-J as shown in the anti-Flag immunoblot of the IP’s (Figure 16A+B). In figure 16b we confirmed the AT14-013 IP with an anti C terminal CD43 antibody. In all samples endogenous CD43 was present, whereas there was only truncated CD43 present up to t F2. We therefore conclude that the epitope of AT14-013 comprises one or more amino acid residues that are present between amino acids 133 and 165. In view of the fact that AT14-013 interacts to a lesser extent with variant F2 (starting at amino acid position 148 as ed in Figure 13), we also conclude that the epitope of AT14-013 at least comprises one or more amino acid residues that are present n amino acids 133 and 147.

Example 5; 13 inhibits AML growth in vivo.

Currently known mental protocols are for instance described in Miller et al., Blood (2013), 1, No.5, e1-e4.

In order to evaluate the cy of AT14-013 against AML in vivo, immunodeficient mice reconstituted with human hematopoietic cells and xenografted with SH-2 cells were treated. Six female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG, The Jackson Laboratory) were humanized by injecting 50 000 CD34+CD38- hematopoietic stem cells in the liver of sublethally irradiated newborns (1-5 days). At 8 weeks, mice were bled to evaluate the engraftment of human hematopoietic cells in their blood. Only mice with higher than % of human chimerism in the peripheral blood were used in this experiment. Five out of 6 mice met this criterion and were intravenously inoculated at d0 with 10x106 SH-2 cells expressing luciferase and GFP. At d14, mice were injected IP with luciferin (150 mg/kg) and the tumor tment was assessed by in vivo bioluminescence. Based on this measurement, mice were randomized in 2 groups and subsequently dosed by iv inoculation of AT14-013 or antibody AT10-002 (against nza, described in , as control) (375 μg) twice per week. The bioluminescence was measured every week as described above. On d39, mice were sacrificed by cervical ation under deep anesthesia and the organs were exposed and fied for bioluminescence. Single-cell suspension was obtained for the liver and the bone marrow and the ce of SH-2 GFP+ cells was quantified by FACS. Treatment of mice engrafted with SH-2 AML cells leads to a tumor growth inhibition of 90.3 % as measured at the sacrifice by whole body measurement (p<0.001, ed ANOVA, Figure 17A).

The number of AML cells, measured by the number of photon per minute (cpm) exhibits a strong decrease in all the organs ed (p=0.0011, ed 2way ANOVA, Figure 17B). This observation is confirmed by the evaluation of the number of tumor cells by FACS in the bone marrow and the liver (p=0.0017, 2way ANOVA, Figure 17C).

Hence, an antibody that is specific for a CD43 peptide described herein is particularly suitable for in vivo treatment or prevention of a myeloproliferative or lymphoproliferative disorder such as AML.

Example 6 Material & Methods Fetal liver, bone marrow and thymus tissue between week 16 and 21 of gestation was obtained from the Human Immune System (HIS) Mouse Facility at the AMC (under Dutch law: Wet Foetaal Weefsel). CD34 enriched mononuclear cell suspensions from tissues were obtained by disrupting whole organs using a Stomacher followed by density gradient centrifugation and magnetic bead separation. CD34 enriched cell suspension of fetal bone marrow was prepared by density gradient centrifugation and magnetic bead separation.

Binding of antibody AT14-013 to cells from fetal liver, fetal thymus and fetal bone marrow was tested by flow cytometry, using commercially ble CD34 (BD, cat. 343516) and CD38 (BD, cat. 303522) antibodies to distinguish the different subsets in these samples.

RESULTS 13 specifically binds to an oncofetal epitope of CD43 As described herein before, AT14-013 is a CD43-specific antibody that recognizes a unique, onco-sialylated tumor antigen that is expressed predominantly by AML and MDS blasts. Tumor antigens are either abnormal proteins with specific expression or ntly expressed normal proteins such as onco-fetal antigens, which are antigens that are normally only expressed during ny by fetal tissues.

Neoplastic transformation of cells is frequently associated with the expression of oncofetal antigens. We found that the AT14-013 epitope of CD43 was expressed by CD34+ CD38- hematopoietic stem cells obtained from fetal liver and fetal bone marrow, but not by CD34+ CD38+ progenitor cells or CD34- CD38- mature cells obtained from fetal liver and fetal bone marrow (Figure 18). These results demonstrate that AT14-013 is able to bind to an oncofetal-sialylated epitope of CD43 that in adults is widely expressed by AML and MDS.

Example 7 AML blasts of donor # 101 (the same donor from whom the B cells producing AT14-013 were obtained) were stained with AT14-013 and with dies specific for CD34 and CD38 (same ure as in Example 6), and with an antibody against CD45 (BD, cat 348815) to distinguish the general blast population (CD45 dim) from healthy cells in the bone marrow and analyzed by flow try (Figure 19). This shows that AT14-013 binds leukemic blasts of the patient it was found in. AT14-013 binds CD34+CD38- blasts that include the leukemic stem cells.

It is therefore concluded that antibody AT14-013 reacts with autologous leukemic stem cells, which makes 13 particularly suitable for treatment or prevention of myeloproliferative or lymphoproliferative disorders e it also targets the ic stem cells, which are known to be more therapy resistant and often responsible for relapse of disease after treatment.

From this it follows that another antibody that is specific for a CD43 e described herein, such as an antibody that competes with antibody AT14-013 for binding to CD43, is also ularly suitable for treatment or prevention of myeloproliferative or lymphoproliferative disorders.

References Bennett, J.M. et al., 1976. Proposals for the classification of the acute leukaemias.

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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 y. ILAR Journal (1995); Vol.37, Number 3: 93-118 International patent application No.

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Immune Network (2014). Vol. 14, No. 3: 0 Kwakkenbos MJ et al. 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 tiis, A. et al., 2011. Mass ometry-Based Identification Of The Tumor Antigen UN1 as the Transmembrane CD43 Sialoglycoprotein. Molecular & Cellular Proteomics, 10(5), 1.007898–M111.007898 Malcovati, L. et al., 2013. Diagnosis and treatment of primary ysplastic syndromes in adults: endations from the European LeukemiaNet. Blood, 122(17), pp.2943–2964 Miller et al., Blood (2013), Vol.121, No.5, e1-e4 Schmid K, Hediger MA, 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 horin (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 Tumours of Haematopoietic and Lymphoid Tissues. ational Agency for ch on , 2008. ISBN: 9788320 Tuccillo et al. Cancer-associated CD43 glycoforms as target of immunotherapy.

Mol.Cancer ther. (2014a) 13(3): 752-762 Tuccillo et al. Aberrant ylation as biomarker for cancer: focus on CD43. BioMed research International (2014b) Article ID 742831, 13 pages. http://dx.doi.org/10.1155/2014/742831 US patent No. 9,005,974

Claims

1. An isolated, recombinant or purified CD43 peptide with a length of at most 100 amino acid es, wherein said peptide comprises an amino acid sequence 5 GTITTNSPETSSRTSGAPVTTAASSLETSRGTS, and wherein said peptide has an acute myeloid leukemia (AML)-specific sialylation pattern or a ysplastic syndrome (MDS)-specific sialylation pattern.

2. A compound, comprising the CD43 peptide according to claim 1.

3. An isolated, synthetic or recombinant nucleic acid molecule encoding the CD43 peptide according to claim 1.

4. A vector comprising the nucleic acid le according to claim 3.

5. Use of the CD43 peptide according to claim 1, the compound according to claim 2, the nucleic acid molecule according to claim 3, or the vector according to claim 4, for inducing, isolating, ing, binding, detecting and/or obtaining an immune cell and/or an antibody, or a functional part thereof that is capable of binding the same antigen as 20 said antibody, directed against said CD43 peptide, wherein said use is not practiced on a living human being.

6. In vitro use of the CD43 peptide according to claim 1, the compound according to claim 2, the nucleic acid molecule according to claim 3, or the vector according to claim 4, 25 for inducing, ing, producing, binding, detecting and/or obtaining an immune cell and/or an antibody, or a functional part thereof that is capable of g the same antigen as said antibody, directed against said CD43 peptide.

7. Use according to claim 5 or 6, wherein said immune cell or antibody or functional 30 part is able to ically bind myeloproliferative or lymphoproliferative cells.

8. The use ing to claim 7, wherein said myeloproliferative cells are acute myeloid leukemia (AML) cells, myelodysplastic syndrome (MDS) cells, or chronic myeloid leukemia (CML) cells.

9. An isolated, recombinant or purified dy, or a functional part thereof that is capable of g the same antigen as said antibody, that competes with an antibody comprising a heavy chain CDR1 sequence SPNWWT and a heavy chain CDR2 sequence EIYYGGRVSYNSALRS and a heavy chain CDR3 sequence AGQKNIGCGYSSCFISWFDT and a light chain CDR1 sequence KSSQTILQRSNHLNYLA and a light chain CDR2 ce WASTRES and a light chain 5 CDR3 sequence HQYYTTPQT for binding to the CD43 peptide according to claim 1 at the GTITTNSPETSSRTSGAPVTTAASSLETSRGTS sequence.

10. An immunogenic composition comprising the CD43 peptide according to claim 1, or the nd according to claim 2, or the nucleic acid le according to claim 3, 10 or the vector ing to claim 4.

11. The immunogenic composition according to claim 10, wherein said composition is a pharmaceutical composition which also comprises a pharmaceutically acceptable carrier, diluent or excipient.

12. A stic kit comprising the CD43 peptide ing to claim 1 or the compound according to claim 2, and means for detecting an antibody or immune cell.

13. Use of the CD43 peptide according to claim 1, or the compound according to claim 20 2, or the nucleic acid molecule according to claim 3, or the vector according to claim 4, in the preparation of a medicament or prophylactic agent for treating or preventing MDS, AML or CML in a patient in need thereof.

14. Use of the CD43 peptide according to claim 1, or a compound according to claim 2, 25 or the nucleic acid le according to claim 3, or the vector according to claim 4, in the preparation of a medicament for treating of a myeloproliferative or lymphoproliferative disorder. 30

15. Use of the CD43 peptide according to claim 1, or the compound according to claim 2, in the preparation of a stic agent.

16. Use of a CD43 peptide according to claim 1, or a compound according to claim 2, in the preparation of a diagnostic agent for diagnosing AML.

17. An in vitro method for determining whether an individual has AML, the method comprising contacting the CD43 peptide ing to claim 1, or the compound according to claim 2, with antibodies and/or immune cells of said individual and determining whether said CD43 peptide or said compound is bound by at least one of said dies 5 and/or immune cells of said individual.

18. A method for producing an immune cell and/or antibody that is able to ically bind lymphoproliferative cells and/or myeloproliferative cells, the method comprising immunizing a non-human animal with the CD43 peptide according to claim 1, or with 10 the nd according to claim 2, or with the nucleic acid molecule according to claim 3, or with the vector according to claim 4.

19. The method according to claim 18, further comprising harvesting an immune cell and/or antibody that is able to specifically bind lymphoproliferative cells and/or 15 myeloproliferative cells, from said non-human animal.

20. An isolated host cell comprising the nucleic acid le according to claim 3, or the vector according to claim 4. 20

21. The method of claim 18 or 19, wherein the immune cell and/or antibody is an AML-specific immune cell or an AML-specific antibody.

22. A CD43 peptide as claimed in claim 1 ntially as herein described and with reference to any example thereof.

23. A compound as claimed in claim 2 substantially as herein described and with reference to any example thereof.

24. A nucleic acid molecule as claimed in claim 3 ntially as herein described 30 and with reference to any example thereof.

25. A vector as claimed in claim 4 substantially as herein described and with reference to any example thereof.

26. A use as claimed in any one of claims 5-8 and 13-16 substantially as herein bed and with reference to any example thereof.

27. An antibody as claimed in claim 9 substantially as herein described and with 5 reference to any example thereof.

28. A composition as claimed in claim 10 or 11 substantially as herein described and with reference to any example thereof. 10

29. A stic kit as claimed in claim 12 substantially as herein described and with reference to any example thereof.

30. A method as claimed in any one of claims 17-19 and 21 substantially as herein described and with reference to any example thereof.

31. A host cell as claimed in claim 20 substantially as herein described and with nce to any example thereof.