and uses thereof

The present invention relates to T cell receptors (TCRs) with specificity for a peptide derived from the protein myeloperoxidase. The present invention further relates to soluble TCR constructs, chimeric TCRs, bi-specific antibodies, nucleic acids, expression constructs and cells comprising said TCRs or TCR constructs. Moreover, the present invention relates to pharmaceutical compositions comprising such TCRs, soluble TCR constructs, chimeric TCRs, bi-specific antibodies, nucleic acids, expression constructs and/or cells. The present invention further relates to the use of such TCRs, soluble TCR constructs, chimeric TCRs, bi-specific antibodies, nucleic acids or expression constructs for generating genetically modified lymphocytes and to their use as medicaments, preferably in the detection, diagnosis, prognosis, prevention and/or treatment of a hematological neoplasia involving the expression of myeloperoxidase, preferably a leukemia involving the expression of myeloperoxidase. The present invention further relates to methods of detecting, diagnosing, prognosing, preventing and/or treating a hematological neoplasia involving the expression of myeloperoxidase, preferably a leukemia involving the expression of myeloperoxidase.

Leukemias, and in particular myeloid neoplasias, are often very aggressive and difficult to treat diseases with high morbidity and mortality. While in recent years significant progress has been made, there is still an urgent need in the art for improved reagents and methods for diagnosing, preventing and treating such leukemias, in particular myeloproliferative neoplasia (MPN), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome and/or myeloproliferative syndrome.

In patients with myeloid leukemia having a high-risk constellation, allogeneic stem cell transplantation is often the only promising treatment option. However, even after allogeneic stem cell transplantation relapse is a common problem. Remarkably, the leukemic cells arising during such relapses can be targeted and often eradicated by T cells. A direct proof of principle of the efficacy of T cells in such treatment situations are the durable remissions induced by the administration of donor lymphocyte infusions to patients with acute and chronic myeloid leukemia (AML and CML) who have relapsed after allogeneic stem cell transplantation (SCT). Such graft versus leukemia (GvL) effects are, however, unpredictable and do not occur in all patients. Moreover, frequently the GvL effects are associated with graft versus host disease (GvHD), and currently little is known about how to target favorable structures for a GvL effect without GvHD.

The advent of technology to redirect T cells to defined leukemia antigens by expression of a transgenic T cell receptor on said T cells offers new opportunities for manipulating the GvL effect, such as by providing leukemia-reactive T cells directed against suitable targets on leukemic cells for patients that lack GvL effects or cannot undergo allogeneic SCT.

It is therefore an object of the present invention to provide for improved means and methods for detecting, preventing and/or treating hematological neoplasia, in particular leukemia, such as myeloproliferative neoplasia (MPN), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplasia syndrome and/or myeloproliferative syndrome, in particular via T cells specific for leukemia antigens or by immunotherapy. In particular, it is an object of the present invention to provide for improved means and methods for preventing/treating relapses in patients after treatment of hematological neoplasia, in particular leukemia (such as myeloproliferative neoplasia (MPN), acute myeloid leukemia (AML), chronic myeloid leukemia (CML)), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome and/or myeloproliferative syndrome, preferably after a treatment involving (allogeneic) stem cell transplantation.

These objects are solved by the below-described aspects of the present invention and the preferable embodiments described.

Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. For the purpose of the present invention, all references cited herein are incorporated by reference in their entireties.

Moreover, the following embodiments can, wherever this does not lead to logical contradictions, be combined with each other without restrictions. Thus, the present disclosure shall encompass, even where not explicitly spelled out in the following, any feasible combination of the embodiments described below. Furthermore, embodiments relating to one aspect of the present invention can, wherever this does not lead to logical contradictions, be combined with another aspect of the present invention without restrictions.

Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of "5 to 50 amino acids" should be interpreted to include not only the explicitly recited values of 5 to 50, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,... 48, 49, 50 and sub-ranges such as from 9 to 50, from5 to 25, from 9 to 25, from 10 to 25, from 10 to 20 and from 15 to 25, etc. This same principle applies to ranges reciting only one numerical value, such as "at least 8 amino acids". Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

A majority of T cells have a T cell receptor (TCR) existing as a complex of several proteins. The actual T cell receptor is composed of two (separate) peptide chains, which are produced from the independent T cell receptor alpha and beta (TCR a and TCR β) genes and are called α- and β- TCR chains, γδ T cells (gamma delta T cells) represent a small subset of T cells that possess a distinct T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR is made up of one γ-chain and one δ-chain. This group of T cells is much less common (2% of total T cells) than the αβ T cells. The structure of the T cell receptor is very similar to immunoglobulin Fab fragments, which are regions defined as the combined light and heavy chain of an antibody arm. Each chain of the TCR is a member of the immunoglobulin superfamily and possesses one N-terminal immunoglobulin (Ig)-variable (V) domain, one Ig-constant (C) domain, a transmembrane/cell membrane-spanning region, and a short cytoplasmic tail at the C-terminal end.

According to the invention, the term "variable region" of the T cell receptor α-chain or β-chain relates to the N-terminal immunoglobulin variable domain of said T cell receptor α-chain or β- chain. The variable domain of both the TCR α-chain and β-chain have three hypervariable or complementarity determining regions (CDR1, CDR2, CDR3), whereas the variable region of the β-chain has an additional area of hypervariability (HV4) that does not normally contact antigen and therefore is not considered a CDR. The three CDRs differ in their function in recognition of Peptide/MHC complexes on the surface of target cells, whereas the CDR3 regions of both chains play the major role in peptide specific recognition of the complex.

According to the invention, the term "constant region" of the T cell receptor α-chain or β-chain relates to the constant domain of the TCR including the transmembrane region and the cytoplasmic tail.

The first signal in activation of T cells is provided by binding of the T cell receptor to a short peptide presented by the major histocompatibility complex (MHC) on another cell. This ensures that only a T cell with a TCR specific to that peptide is activated.

Hematological neoplasia cells like leukemia cells, in particular myeloproliferative neoplasia (MPN) cells, acute myeloid leukemia (AML) cells, chronic myeloid leukemia (CML) cells, acute lymphoblastic leukemia (ALL) cells, myelodysplasia syndrome cells and myeloproliferative syndrome cells, express, like many other tumors, tumor-associated antigens. Myeloperoxidase (MPO) is a differentiation antigen with restricted expression in myeloid precursors, mature neutrophils, monocytes and macrophages, but otherwise not in healthy human cells outside of the embryo stage. Thus, MPO can serve as a specific marker for hematological neoplasia cells showing expression of myeloperoxidase (i.e. MPO-positive hematological neoplasia cells). In particular, as a myeloid differentiation antigen, MPO can serve as a specific marker for myeloid leukemias, such as in myeloproliferative neoplasia (MPN) cells, acute myeloid leukemia (AML) cells, chronic myeloid leukemia (CML) cells, myelodysplastic syndrome cells and myeloproliferative syndrome cells. Moreover, MPO can serve as a specific marker for certain acute lymphoblastic leukemias (ALL) showing expression of myeloperoxidase.

Whether hematological neoplasia cells or leukemia cells of a certain type of leukemia express myeloperoxidase at the protein level can be determined by the skilled person by validated standard methods, such as immunohistochemistry on bone marrow smears or intracellular staining of fixed cells with a myeloperoxidase-specific antibody followed by flow cytometric analysis, preferably by immunohistochemistry on bone marrow smears. Healthy myeloid cells can be equally detected by antibody staining as flow cytometry and immunohistochemistry of blood and bone marrow. Furthermore, whether hematological neoplasia cells or leukemia cells of a certain type of leukemia express myeloperoxidase at the mRNA level can be detected by qRT- PCR or RNA in situ hybridisation, preferably by RNA in situ hybridisation.

T cell receptor

In a first aspect, the present invention relates to a T cell receptor (TCR) or a fragment thereof comprising:

(i) a T cell receptor cc-chain or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 3 (CDR3 of T cell receptor a-chain) or an amino acid sequence that has at least 75% identity, more preferably at least 83% identity, more preferably at least 91% identity with the amino acid sequence of SEQ ID NO: 3;

and

(ii) a T cell receptor β-chain or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 6 (CDR3 of T cell receptor β-chain) or an amino acid sequence that has at least 76% identity, more preferably at least 84% identity, more preferably at least 92% identity with the amino acid sequence of SEQ ID NO: 6.

In some embodiments, said T cell receptor or said fragment thereof is capable of binding to a peptide having the amino acid sequence of SEQ ID NO: 7 (MPO466-474 peptide) bound to HLA- B*07:02 (Human Leukocyte Antigen, allele B* 07:02) and/or to a peptide having the amino acid sequence of SEQ ID NO: 7 bound to HLA-B *O7:0¥ and/or to a peptide having the amino acid sequence of SEQ ID NO: 7 bound to ELA-B *42:01, more preferably to a peptide having the amino acid sequence of SEQ ID NO: 7 bound to HLA-B *07:02.

In some embodiments, said fragment of said T cell receptor α-chain comprises at least the variable region of said T cell receptor α-chain or comprises at least a sequence of 25, preferably at least a sequence of 50, more preferably at least a sequence of 100 amino acids of said T cell receptor α-chain.

In some embodiments, said fragment of said T cell receptor β-chain comprises at least the variable region of said T cell receptor β-chain or comprises at least a sequence of 25, preferably at least a sequence of 50, more preferably at least a sequence of 100 amino acids of said T cell receptor β-chain.

In view of sequence conservation of these regions between different T cell receptor families, the CDR1 and CDR2 regions play, compared to CDR3, only an auxiliary role for the peptide specificity of the T cell receptor.

In some embodiments, said T cell receptor α-chain or said fragment thereof further comprises

- the amino acid sequence of SEQ ID NO: 1 (CDR1 of T cell receptor a-chain) or an amino acid sequence that has at least 83% identity with the amino acid sequence of SEQ ID NO: 1,

and/or

- the amino acid sequence of SEQ ID NO: 2 (CDR2 of T cell receptor a-chain) or an amino acid sequence that has at least 75% identity, more preferably at least 87% identity with the amino acid sequence of SEQ ID NO: 2.

In some embodiments, said T cell receptor β-chain or said fragment thereof further comprises

- the amino acid sequence of SEQ ID NO: 4 (CDR1 of T cell receptor β-chain) or an amino acid sequence that has at least 80% identity with the amino acid sequence of SEQ ID NO: 4, and/or

- the amino acid sequence of SEQ ID NO: 5 (CDR2 of T cell receptor β-chain) or an amino acid sequence that has at least 83% identity with the amino acid sequence of SEQ ID NO: 5.

In some embodiments, all amino acid exchanges by which said amino acid sequence that has at least 75% identity, more preferably at least 83% identity, more preferably at least 91% identity with the amino acid sequence of SEQ ID NO: 3 differs from the amino acid sequence of SEQ ID NO: 3 are conservative amino acid exchanges.

In some embodiments, all amino acid exchanges by which said amino acid sequence that has at least 76% identity, more preferably at least 84% identity, more preferably at least 92% identity with the amino acid sequence of SEQ ID NO: 6 differs from the amino acid sequence of SEQ ID NO: 6 are conservative amino acid exchanges.

In some embodiments, all amino acid exchanges by which said amino acid sequence that has at least 83% identity with the amino acid sequence of SEQ ID NO: 1 differs from the amino acid sequence of SEQ ID NO: 1 are conservative amino acid exchanges.

In some embodiments, all amino acid exchanges by which said amino acid sequence that has at least 75% identity, more preferably at least 87% identity with the amino acid sequence of SEQ ID NO: 2 differs from the amino acid sequence of SEQ ID NO: 2 are conservative amino acid exchanges.

In some embodiments, all amino acid exchanges by which said amino acid sequence that has at least 80% identity with the amino acid sequence of SEQ ID NO: 4 differs from the amino acid sequence of SEQ ID NO: 4 are conservative amino acid exchanges.

In some embodiments, all amino acid exchanges by which said amino acid sequence that has at least 83% identity with the amino acid sequence of SEQ ID NO: 5 differs from the amino acid sequence of SEQ ID NO: 5 are conservative amino acid exchanges. In some embodiments, said T cell receptor α-chain (or said fragment thereof) is covalently linked to said T cell receptor β-chain (or said fragment thereof), preferably by a peptide bond. In some embodiments, said T cell receptor α-chain (or said fragment thereof) and said T cell receptor β- chain (or said fragment thereof) are part of the same polypeptide chain.

In some embodiments, said T cell receptor α-chain (or said fragment thereof) and said T cell receptor β-chain (or said fragment thereof) are not part of the same polypeptide chain. In some embodiments said T cell receptor α-chain (or said fragment thereof) is not covalently linked to said T cell receptor β-chain (or said fragment thereof).

Preferably, the part of said T cell receptor α-chain (or said fragment thereof) having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that has at least 83% identity with the amino acid sequence of SEQ ID NO: 1 forms the CDR1 (Complementarity Determining Region 1) region of said T cell receptor α-chain (or said fragment thereof).

Preferably, the part of said T cell receptor α-chain (or said fragment thereof) having the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that has at least 75% identity, more preferably at least 87% identity with the amino acid sequence of SEQ ID NO: 2 forms the CDR2 (Complementarity Determining Region 2) region of said T cell receptor α-chain (or said fragment thereof).

Preferably, the part of said T cell receptor α-chain (or said fragment thereof) having the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that has at least 75% identity, more preferably at least 83% identity, more preferably at least 91% identity with the amino acid sequence of SEQ ID NO: 3 forms the CDR3 (Complementarity Determining Region 3) region of said T cell receptor α-chain (or said fragment thereof).

Preferably, the part of said T cell receptor β-chain (or said fragment thereof) having the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that has at least 80% identity with the amino acid sequence of SEQ ID NO: 4 forms the CDR1 (Complementarity Determining Region 1) region of said T cell receptor β-chain (or said fragment thereof).

Preferably, the part of said T cell receptor β-chain (or said fragment thereof) having the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence that has at least 83% identity with the amino acid sequence of SEQ ID NO: 5 forms the CDR2 (Complementarity Determining Region 2) region of said T cell receptor β-chain (or said fragment thereof).

Preferably, the part of said T cell receptor β-chain (or said fragment thereof) having the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence that has at least 76% identity, more preferably at least 84% identity, more preferably at least 92% identity with the amino acid sequence of SEQ ID NO: 6 forms the CDR3 (Complementarity Determining Region 3) region of said T cell receptor β-chain (or said fragment thereof).

In some embodiments, the variable region of said T cell receptor α-chain or said fragment thereof is or comprises Val.l and/or the variable region of said T cell receptor β-chain or said fragment thereof is or comprises νβ 13.2

In some embodiments,

a) said T cell receptor α-chain (or said fragment thereof) and/or said T cell receptor β- chain (or said fragment thereof) comprise or are composed of human sequences or b) said T cell receptor α-chain (or said fragment thereof) and/or said T cell receptor β- chain (or said fragment thereof) comprise or are composed of mouse sequences or c) said T cell receptor α-chain (or said fragment thereof) and/or said T cell receptor β- chain (or said fragment thereof) are each composed of a combination of human and mouse sequences, wherein, preferably, the constant region of said T cell receptor α- chain and the constant region of said T cell receptor β-chain are composed of mouse sequences and, preferably, the variable region of said T cell receptor α-chain and the variable region of said T cell receptor β-chain are composed of human sequences.

If a T cell receptor is used in human cells which T cell receptor has an α-chain and/or β-chain having a constant region composed of mouse sequences, this has the advantage that mispairing of said T cell receptor α-chain and/or β-chain with endogenous T cell receptor chains of said human cells is reduced/avoided.

In some embodiments, said T cell receptor (or said fragment thereof) comprises two disulfide bonds. Preferably, said T cell receptor α-chain (or said fragment thereof) and said T cell receptor β-chain (or said fragment thereof) are linked by said two disulfide bonds. This has the technical effect that mispairing of transgenic chains with endogenous chains is reduced/avoided.

In some embodiments, said T cell receptor comprises

(i) a T cell receptor α-chain having an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 8 (full-length sequence of a-chain of T cell receptor TCR2.5D6) or an amino acid sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the amino acid sequence of SEQ ID NO: 8,

and/or

(ii) a T cell receptor β-chain having an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 9 (full-length sequence of β-chain of T cell receptor TCR2.5D6) or an amino acid sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the amino acid sequence of SEQ ID NO: 9.

In some embodiments, said fragment of said T cell receptor comprises

(i) a fragment of a T cell receptor α-chain having the amino acid sequence of SEQ ID NO: 8 (full-length sequence of a-chain of T cell receptor TCR2.5D6) or of an amino acid sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the amino acid sequence of SEQ ID NO: 8,

wherein, preferably, said fragment comprises at least the variable region of said T cell receptor α-chain having the amino acid sequence of SEQ ID NO: 8 (or of said amino acid sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the amino acid sequence of SEQ ID NO: 8) or comprises at least a sequence of 25, preferably at least a sequence of 50, more preferably at least a sequence of 100 amino acids of said T cell receptor α-chain having the amino acid sequence of SEQ ID NO: 8 (or of said amino acid sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the amino acid sequence of SEQ ID NO: 8);

and/or

(ii) a fragment of a T cell receptor β-chain having the amino acid sequence of SEQ ID NO: 9 (full-length sequence of β-chain of T cell receptor TCR2.5D6) or of an amino acid sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the amino acid sequence of SEQ ID NO: 9,

wherein, preferably, said fragment comprises at least the variable region of said T cell receptor β-chain having the amino acid sequence of SEQ ID NO: 9 (or of said amino acid sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the amino acid sequence of SEQ ID NO: 9) or comprises at least a sequence of 25, preferably at least a sequence of 50, more preferably at least a sequence of 100 amino acids of said T cell receptor β-chain having the amino acid sequence of SEQ ID NO: 9 (or of said amino acid sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the amino acid sequence of SEQ ID NO: 9).

In some embodiments, said T cell receptor or said fragment thereof is a free molecule. In some embodiments, said T cell receptor or said fragment thereof is not linked to another molecule by covalent linkage. In some embodiments, said T cell receptor or said fragment thereof is not linked to another molecule. In some embodiments, said T cell receptor or said fragment thereof is not immobilized on a surface. In some embodiments, said T cell receptor or said fragment thereof is not immobilized on a cell.

In some embodiments, said T cell receptor or said fragment thereof is linked to another molecule, preferably by covalent linkage. In some embodiments, said T cell receptor or said fragment thereof is immobilized on a surface. In some embodiments, said T cell receptor or said fragment thereof is immobilized on a cell.

Soluble T cell receptor construct

In a second aspect, the present invention relates to a soluble T cell receptor (sTCR) construct comprising

(1) a T cell receptor component comprising

at least one T cell receptor α-chain (or fragment thereof) as defined in any of the embodiments above and

at least one T cell receptor β-chain (or fragment thereof) as defined in any of the embodiments above,

preferably covalently linked to each other to form a TCR heterodimer or multimer, and

(2) fusion component(s)

preferably selected from

- an Fc receptor,

- an Fc domain of an antibody,

- a cytokine, preferably IL-2 or IL- 15 ,

- a toxin,

- an antibody, preferably selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD5 antibody, an anti-CD 16 antibody and an anti-CD56 antibody,

- a linker specific chimeric antigen receptor (CAR) expressed in a T cell, wherein the CAR recognizes a linker that is attached to the soluble T cell receptor construct, or combinations thereof, wherein said at least one T cell receptor α-chain (or said fragment thereof) of (1) and/or said at least one T cell receptor β-chain (or that fragment thereof) of (1) is bound to the fusion component(s) of (2),

and wherein, preferably, the soluble T cell receptor construct furthermore comprises a label, preferably selected from the group consisting of a radionuclide, a fluorophore and gold particles.

Preferably, said fluorophore is fluorescein.

Preferably, said toxin is an exotoxin, more preferably pseudomonas exotoxin.

The soluble TCRs can comprise different of the fusion components (2), such as cytokines and Fc domains. TCR α-chains and TCR β-chains (or fragments thereof) can be solubilized by chemical modifications and linked covalently to each other and to other molecules such as Fc-receptors, cytokines and toxins (as described in Boulter et al, 2005; Lunde et al., 2010). These soluble TCR constructs are suitable to be directly used for the detection and destruction of MPO-positive hematological neoplasia cells, such as MPO-positive leukemia cells, for example myeloproliferative neoplasia (MPN) cells, the cells of an MPO-positive acute leukemia (such as acute myeloid leukemia (AML) cells), the cells of an MPO-positive chronic leukemia (such as chronic myeloid leukemia (CML) cells), the cells of an MPO-positive acute lymphoblastic leukemia (ALL), myelodysplastic syndrome cells or myeloproliferative syndrome cells, without the need of T cells. This can be achieved by application of soluble T cell receptor constructs to patients by intravenous, subcutaneous or intramuscular infusion or injections, respectively, or by application to the mucosa of the respiratory tract by inhalation or by spraying.

Linker specific chimeric antigen receptors (CARs) are described in Urbanska K et al., 2012 and Koristka S et al., 2014. A linker (e.g. biotin or a peptide derived from a nuclear protein) can be attached to the soluble TCRs. T cells transgenic for a CAR recognizing the linker (i.e. a linker specific CAR) can thereby be redirected to MPO-positive hematological neoplasia cells, such as MPO-positive leukemia cells, for example myeloproliferative neoplasia (MPN) cells, the cells of an MPO-positive acute leukemia (such as acute myeloid leukemia (AML) cells), the cells of an MPO-positive chronic leukemia (such as chronic myeloid leukemia (CML) cells), the cells of an MPO-positive acute lymphoblastic leukemia (ALL), myelodysplastic syndrome cells or myeloproliferative syndrome cells.

In some embodiments, the soluble TCR constructs of the present invention are used as recombinant proteins. Such recombinant proteins are expressed, preferably in E.coli or mammalian cells, and then purified before further use or application.

The present invention provides the expressed and purified soluble TCR constructs.

Chimeric T cell receptor

In a third aspect, the present invention relates to a chimeric T cell receptor or fragment thereof comprising at least one T cell receptor or fragment thereof according to any of the embodiments above, wherein the T cell receptor α-chain (or fragment thereof) and/or the T cell receptor β- chain (or fragment thereof) is/are fused, preferably via a linker, to CD3-zeta chain(s) and/or one or more other T cell receptor stimulation domains, preferably selected from the group consisting of the intracellular CD28, CD137 or CD134 domains.

In some embodiments, said T cell receptor α-chain (or said fragment thereof) and said T cell receptor β-chain (or said fragment thereof) of said chimeric TCR are fused to each other via a linker.

Preferred CD3-zeta chain(s) are the zetα-chain(s) of the human CD3 complex of the T cell receptor. Preferred CD28, CD 137 and CD 134 intracellular domains are of human origin.

The chimeric TCRs or fragments thereof can comprise further components.

Chimeric T cell receptors and chimeric antigen receptors (CAR) are known in the art. See, for example, Han et al., 2013. Bi-specific antibody

In a fourth aspect, the present invention relates to a bi-specific antibody comprising

(a) a T cell receptor component comprising the T cell receptor α-chain (or fragment thereof) and the T cell receptor β-chain (or fragment thereof) of a T cell receptor (or fragment thereof) according to any of the embodiments above,

wherein said T cell receptor α-chain (or said fragment thereof) and said T cell receptor β-chain (or said fragment thereof) are linked to each other

and fused, preferably via a linker, to

(b) an antibody or a single chain antibody fragment (scFv) or an linker specific chimeric antigen receptor (CAR), preferably and antibody, which is directed against an antigen or epitope on the surface of lymphocytes.

In some embodiments, said T cell receptor α-chain (or said fragment thereof) and said T cell receptor β-chain (or said fragment thereof) are linked to each other covalently.

In some embodiments, the antibody or scFv or linker specific chimeric antigen receptor (CAR) is/are directed against CD3 or CD5 on the surface of T cells. In this embodiment, the bi-specific antibody will bind to T cells and redirect them to myeloperoxidase-presenting tumor cells (i.e. to tumor cells which present the peptide corresponding to myeloperoxidase, residues 466-474, bound to HLA-B*07:02/B*07:4/B*42:01 at their cell surface).

In another embodiment, the antibody or scFv or linker specific chimeric antigen receptor (CAR) is/are directed against the co-stimulatory CD28 receptor on T cells. In this embodiment, the bi- specific antibody will bind to and co-activate T cells and direct them to myeloperoxidase- presenting tumor cells.

In another embodiment, antibodies or scFV or linker specific chimeric antigen receptors (CARs) against CD3 or CD5 and CD28 are combined in tetravalent antibody constructs. In some embodiments, the antibody or scFv or linker specific chimeric antigen receptor (CAR) is/are directed against the CD56 receptor of NK cells. In this embodiment, the bi-specific antibody will bind to NK cells and redirect them to myeloperoxidase-presenting tumor cells.

In another embodiment, the antibody or scFv or linker specific chimeric antigen receptor (CAR) is/are directed against the activating receptor CD 16 on NK cells. In this embodiment, the bi- specific antibody will bind to and activate NK cells and direct them to myeloperoxidase- presenting tumor cells.

In another embodiment, antibodies or scFV or linker specific chimeric antigen receptors (CARs) against CD56 and CD 16 are combined in tetravalent antibody constructs.

For example, a bi-specific antibody can be a bi-specific T cell engager (BiTE) which is a class of artificial bi-specific monoclonal antibodies that are investigated for their use as anti-cancer drugs. They direct a host's immune system, more specifically the T cells' cytotoxic activity, against cancer cells. BiTE is a registered trademark of Micromet AG. BiTEs are fusion proteins consisting of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of about 55 kilodaltons. One of the scFvs binds to T cells such as via the CD3 receptor or to CD56 for NK cell activation, and the other to a tumor cell via a tumor-specific molecule (see e.g. Csoka et al., 1996).

Preferably, the bi-specific antibodies of the present invention are used as recombinant proteins. Such recombinant proteins are expressed, preferably in E.coli or mammalian cells, and then purified before further use or application.

The present invention provides the expressed and purified bi-specific antibodies. Nucleic acid/Set of nucleic acids

In a fifth aspect, the present invention relates to a nucleic acid encoding

- the amino acid sequence of a T cell receptor or fragment thereof according to any of the embodiments above,

- the amino acid sequence of a soluble T cell receptor construct according to any of the embodiments above,

- the amino acid sequence of a chimeric T cell receptor or fragment thereof according to any of the embodiments above, or

- the amino acid sequence of the T cell receptor component of a bi-specific antibody according to any of the embodiments above;

or

- a set of nucleic acids, one of which encodes the amino acid sequence of the T cell receptor α- chain (or fragment thereof) and one of which the amino acid sequence of the T cell receptor β- chain (or fragment thereof) of a T cell receptor or fragment thereof according to any of the embodiments above;

- a set of nucleic acids, one of which encodes the amino acid sequence of the T cell receptor α- chain (or fragment thereof) and one of which the amino acid sequence of the T cell receptor β- chain (or fragment thereof) of a soluble T cell receptor construct as defined above;

- a set of nucleic acids, one of which encodes the amino acid sequence of the T cell receptor α- chain (or fragment thereof) and one of which the amino acid sequence of the T cell receptor β- chain (or fragment thereof) of a chimeric T cell receptor or fragment thereof as defined above; or

- a set of nucleic acids, one of which encodes the amino acid sequence of the T cell receptor α- chain (or fragment thereof) and one of which the amino acid sequence of the T cell receptor β- chain (or fragment thereof) of the T cell receptor component of a bi-specific antibody as defined above.

The nucleic acids according to this invention comprise DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA), combinations thereof or derivatives (such as PNA) thereof. Preferably, said nucleotide sequence is a DNA sequence. Preferably, said set of nucleotide sequences is a set of DNA sequences.

Codon-optimization refers to the exchange in a sequence of interest of codons that are generally rare in highly expressed genes of a given species by codons that are generally frequent in highly expressed genes of such species, such codons encoding the same amino acids as the codons that are being exchanged. Preferably, the nucleic acid sequences of the present invention are codon- optimized for expression in mammalian cells, preferably for expression in human cells, or comprise sequence stretches that are codon-optimized for expression in mammalian cells, preferably for expression in human cells. For generation of bi-specific antibodies, the nucleic acid sequences can also be codon-optimized for expression in bacteria, yeast or insect cells.

Within the scope of this invention are also the nucleotide sequences obtained due to the degeneration of the genetic code of the above nucleotide sequences.

Preferably, said nucleic acid or set of nucleic acids comprises

- the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11 or a nucleotide sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11,

or a nucleotide sequence that is complementary to such a sequence;

and/or

- the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13 or a nucleotide sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13,

or a nucleotide sequence that is complementary to such a sequence. Preferably, said nucleic acid or set of nucleic acids comprises

a fragment of the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11 or of a nucleotide sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11,

or a fragment of a nucleotide sequence that is complementary to such a sequence, wherein, preferably, said fragment encodes at least the variable region of the T cell receptor α-chain amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11 (or of an amino acid sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11) or comprises at least a sequence of 30, preferably at least a sequence of 75, more preferably at least a sequence of 150 nucleotides of the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11 or of a nucleotide sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity with a nucleotide sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11;

and/or

a fragment of the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13 or of a nucleotide sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13,

or a fragment of a nucleotide sequence that is complementary to such a sequence, wherein, preferably, said fragment encodes at least the variable region of the T cell receptor ct-chain amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13 (or of an amino acid sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13) or comprises at least a sequence of 30, preferably at least a sequence of 75, more preferably at least a sequence of 150 nucleotides of the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13 or of a nucleotide sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity with a nucleotide sequence that has at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13.

The nucleotide sequences of SEQ ID NO. 10 to 13 are codon-optimized for expression in human cells.

Expression construct

In a sixth aspect, the present invention relates to an expression construct for expressing the T cell receptor or fragment thereof according to any of the embodiments above, the soluble T cell receptor construct according to any of the embodiments above, the chimeric T cell receptor or fragment thereof according to any of the embodiments above or the bi-specific antibody according to any of the embodiments above in a host cell.

Preferably, said expression construct comprises a DNA sequence encoding

- the amino acid sequence of a T cell receptor or fragment thereof according to any of the embodiments above, - the amino acid sequence of a soluble T cell receptor construct according to any of the embodiments above,

- the amino acid sequence of a chimeric T cell receptor or fragment thereof according to any of the embodiments above, or

- the amino acid sequence of the T cell receptor component of a bi-specific antibody according to any of the embodiments above.

Preferably, said expression construct comprises promoter and terminator sequences.

Preferably, said host cell is a mammalian cell or a bacterial cell, more preferably a human cell. Preferably, said host cell is a cultured cell.

An "expression construct" or "gene construct" (wherein both terms are used interchangeably throughout this specification) refers to a nucleic acid construct, usually an expression vector or plasmid that is used to introduce a specific gene sequence into a target cell (the host cell). Once the expression or gene construct is inside the host cell, the protein that is encoded by the gene is produced by the cellular transcription and translation machinery. The expression or gene construct is designed to contain respective regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the construct, including promoter and terminator sequences. The goal of a well-designed expression or gene construct is the production of large amounts of stable mRNA, and therefore proteins. Alternatively, an RNA is synthesized or in vitro transcribed and introduced directly into the host cells for protein expression.

The skilled artisan can select further suitable components of expression or gene constructs. Examples for suitable expression constructs are viral vectors, such as

- retroviral vectors, such as MP71 vectors (as described in Engels et al., 2003), or retroviral SIN vectors (as described in Yu et al., 1986);

and

- lentiviral vectors or lentiviral SIN vectors, as described in Miyoshi et al., 1998. Viral vectors containing TCR-encoding genes can infect lymphocytes and express functional TCR in infected lymphocytes. Preferred is a SIN vector construct.

Another example for a suitable vector is the Sleeping Beauty (SB) transposon transposase DNA plasmid system, SB DNA plasmid (as described in Maiti et al, 2013). Further options are the vector of the piggyBac transposition system (Manuri PV et al., 2010) or vectors for chromosomal insertion through the CRISPR/CAS9 system (Mandal PK et al., 2014).

The nucleic acids and/or in particular expression constructs of the invention can also be transferred into host cells by transient RNA transfection.

The nucleic acids and/or in particular expression constructs of the invention are capable of directing the synthesis/expression of the TCRs of the invention (or fragments thereof) in a suitable host cell.

Cell

In a seventh aspect, the present invention relates to a cell comprising the T cell receptor (or fragment thereof) according to any of the embodiments above, the soluble T cell receptor construct according to any of the embodiments above, the chimeric T cell receptor (or fragment thereof) according to any of the embodiments above, the bi-specific antibody according to any of the embodiments above, the nucleic acid or set of nucleic acids according to any of the embodiments above or the expression construct according to any of the embodiments above.

As used herein, the term "cell" refers to a eukaryotic cell (for example a mammalian cell or yeast cell) or prokaryotic cell (for example a bacterial cell, such as an E.coli cell). Thus, a cell is a structural unit of living organisms, which is capable of self replicating, has genetic information and a mechanism for expressing it, and is surrounded by a membrane structure which isolates the cell from the surrounding environment.

Preferably, said cell is a transgenic Preferably, said cell comprises a transgene, wherein said transgene comprises said nucleic acid or set of nucleic acids according to any of the embodiments above.

As used herein, the term "transgenic cell" refers to a cell that has a transgene stably integrated into its genome and that transmits the transgene to its progeny during cell division. As used herein, the term "transgene" refers to a heterologous gene that is stably integrated into the genome of a cell and that is transmitted to progeny of the cell during cell division.

Preferably, said cell expresses the T cell receptor (or fragment thereof) according to the present invention. Preferably, said cell expresses the soluble T cell receptor construct according to the present invention. Preferably, said cell expresses the chimeric T cell receptor (or fragment thereof) according to the present invention. Preferably, said cell expresses the bi-specific antibody according to the present invention.

Preferably, said cell is a cell for expression of the T cell receptor (or fragment thereof) according to the present invention. Preferably, said cell is a cell for expression of the soluble T cell receptor construct according to the present invention. Preferably, said cell is a cell for expression of the chimeric T cell receptor (or fragment thereof) according to the present invention. Preferably, said cell is a cell for expression of the bi-specific antibody according to the present invention.

Preferably, said cell expresses the T cell receptor (or fragment thereof) according to any of the embodiments above, the soluble T cell receptor construct according to any of the embodiments above, the chimeric T cell receptor (or fragment thereof) according to any of the embodiments above or the bi-specific antibody according to any of the embodiments above.

Preferably, said cell is a mammalian cell, more preferably a human cell.

Preferably, said cell is a cultured cell or a cell isolated from the organism that it originates from.

Preferably, said cell is a lymphocyte (preferably a T cell or a natural killer (NK) cell). More preferably, said cell is a T cell, even more preferably a CD3 ⁺ T cell or CD8 ⁺ T cell. The transfer of myeloperoxidase-specific TCR genes into (primary) T cells by e.g. (retro-)viral vectors, SB DNA or by transient RNA transfection represents a promising means and method to generate myeloperoxidase antigen-specific T cells. Adoptive transfer of such engineered T cells into hosts represents a promising approach for the immunotherapy of MPO-positive hematological neoplasia, such as MPO-positive leukemia, for example myeloproliferative neoplasia (MPN), MPO-positive acute leukemia (such as acute myeloid leukemia (AML)), MPO-positive chronic leukemia (such as chronic myeloid leukemia (CML)), MPO-positive acute lymphoblastic leukemia (ALL), myelodysplastic syndrome or myeloproliferative syndrome.

Preferably, said cell for expression of the T cell receptor (or fragment thereof) according to the present invention or for expression of the bi-specific antibody according to the present invention is selected from the group consisting of an E. coli cell, an insect cell and a mammalian cell. As the skilled person will appreciate, it is advantageous to use cells that express high amounts of recombinant protein. Thus, among mammalian cells HEK293 cells are preferred.

The application of myeloperoxidase-specific bi-specific antibodies or soluble T cell receptors represents an alternative promising means and method to target myeloperoxidase-positive tumor cells by redirected T cells or by the soluble T cell receptor construct. This represents a promising approach for the immunotherapy of leukemia, in particular myeloproliferative neoplasia (MPN), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML).

Pharmaceutical composition

In an eighth aspect, the present invention relates to a pharmaceutical composition comprising one or more of:

(i) the T cell receptor (or fragment thereof) according to any of the embodiments above;

(ii) the soluble T cell receptor construct according to any of the embodiments above;

(iii) the chimeric T cell receptor (or fragment thereof) according to any of the embodiments above;

(iv) the bi-specific antibody according to any of the embodiments above; (v) the nucleic acid or set of nucleic acids according to any of the embodiments above or the expression construct according to any of the embodiments above; and/or

(vi) the cell according to any of the embodiments above,

and, optionally, pharmaceutical excipient(s).

Use for generating genetically modified lymphocytes

In a ninth aspect, the present invention relates to the use of a T cell receptor (or fragment thereof) according to any of the embodiments above, a chimeric T cell receptor (or fragment thereof) according to any of the embodiments above, a bi-specific antibody according to any of the embodiments above, a nucleic acid or set of nucleic acids according to any of the embodiments above or an expression construct according to any of the embodiments above for generating genetically modified lymphocytes (preferably selected from the group consisting of T cells, NK cells and NKT cells), preferably genetically modified T cells, more preferably genetically modified CD3 ⁺ T cells or CD8 ⁺ T cells.

Preferably, said use is an in vitro use or an in vivo use.

In some embodiments, the generated genetically modified lymphocytes, preferably T cells and/or NK cells, are for use in immunotherapy or donor lymphocyte infusions or purging in the context of induction or consolidation therapy with autologous modified cells, preferably for preventing and/or treating MPO-positive hematological neoplasia, preferably MPO-positive leukemia, more preferably a disease selected from the group consisting of myeloproliferative neoplasia (MPN), MPO-positive acute leukemia (preferably acute myeloid leukemia (AML)), MPO-positive chronic leukemia (preferably chronic myeloid leukemia (CML)), MPO-positive acute lymphoblastic leukemia (ALL), myelodysplasia syndrome and myeloproliferative syndrome, even more preferably acute myeloid leukemia (AML).

The transfer of myeloperoxidase-specific TCR genes (or parts thereof) into (primary) T cells by e.g. (retro-)viral vectors or by transient RNA transfection represents a promising means and method to generate myeloperoxidase antigen- specific T cells. Adoptive transfer of such engineered T cells into hosts represents a promising approach for the immunotherapy of MPO- positive hematological neoplasia, such as MPO-positive leukemia, for example of myeloproliferative neoplasia (MPN), MPO-positive acute leukemia (e.g. acute myeloid leukemia (AML)), MPO-positive chronic leukemia (e.g. chronic myeloid leukemia (CML)), MPO-positive acute lymphoblastic leukemia (ALL), myelodysplastic syndrome and myeloproliferative syndrome. A new therapeutic approach is the adoptive T cell therapy. The transferred T cells can recognize tumor ' antigen and thereby specifically eliminate tumor cells. MPO-positive hematological neoplasia, such as MPO-positive leukemia, for example of myeloproliferative neoplasia (MPN), MPO-positive acute leukemia (e.g. acute myeloid leukemia (AML)), MPO- positive chronic leukemia (e.g. chronic myeloid leukemia (CML)), MPO-positive acute lymphoblastic leukemia (ALL), myelodysplastic syndrome and myeloproliferative syndrome, are targets for T cell therapy because such tumors express, like many other tumors, tumor-associated antigens, such as myeloperoxidase.

The transfer of chimeric receptors containing the whole or parts of the myeloperoxidase-specific TCR genes linked to the CD3zeta chain into primary NK cells by e.g. (retro-)viral vectors or by transient RNA transfection represents a promising means and method to generate myeloperoxidase antigen-specific NK cells. Adoptive transfer of such engineered NK cells into hosts represents a promising approach for the immunotherapy of MPO-positive hematological neoplasia, such as MPO-positive leukemia, for example of myeloproliferative neoplasia (MPN), MPO-positive acute leukemia (e.g. acute myeloid leukemia (AML)), MPO-positive chronic leukemia (e.g. chronic myeloid leukemia (CML)), MPO-positive acute lymphoblastic leukemia (ALL), myelodysplastic syndrome and myeloproliferative syndrome.

Said genetically modified lymphocytes may be used for adoptive, target cell specific immunotherapy or genetic immunization of a patient. Preferably, said patient suffers from an MPO-positive hematological neoplasia, preferably from MPO-positive leukemia, more preferably from a disease selected from the group consisting of myeloproliferative neoplasia (MPN), MPO-positive acute leukemia (preferably acute myeloid leukemia (AML)), MPO- positive chronic leukemia (preferably chronic myeloid leukemia (CML)), MPO-positive acute lymphoblastic leukemia (ALL), myelodysplastic syndrome and myeloproliferative syndrome, even more preferably from acute myeloid leukemia (AML). Preferably, said patient is HLA- B*07:02 positive and/or HLA-B*07:04 positive and/or HLA-B*42:01 positive, more preferably HLA-B*07: 02 positive.

Medical uses

With respect to the different aspects of the present invention described above, in some embodiments the T cell receptor (or fragment thereof) according to any of the embodiments above, the soluble T cell receptor construct according to any of the embodiments above, the chimeric T cell receptor (or fragment thereof) according to any of the embodiments above, the bi-specific antibody according to any of the embodiments above, the nucleic acid or set of nucleic acids according to any of the embodiments above, the expression construct according to any of the embodiments above, the cell according to any of the embodiments above or the pharmaceutical composition as defined above is for use as a medicament.

With respect to the different aspects of the present invention described above, in some embodiments the T cell receptor (or fragment thereof) according to any of the embodiments above, the soluble T cell receptor construct according to any of the embodiments above, the chimeric T cell receptor (or fragment thereof) according to any of the embodiments above, the bi-specific antibody according to any of the embodiments above, the nucleic acid or set of nucleic acids according to any of the embodiments above, the expression construct according to any of the embodiments above, the cell according to any of the embodiments above or the pharmaceutical composition as defined above is for use in the detection, diagnosis or prognosis of a hematological neoplasia, preferably of leukemia, more preferably of a disease selected from the group consisting of myeloproliferative neoplasia (MPN), acute leukemia (preferably acute myeloid leukemia (AML)), chrome leukemia (preferably chronic myeloid leukemia (CML)), acute lymphoblastic leukemia (ALL), myelodysplasia syndrome and myeloproliferative syndrome, even more preferably of acute myeloid leukemia (AML).

Preferably, said hematological neoplasia is an MPO-positive hematological neoplasia, preferably MPO-positive leukemia, more preferably a disease selected from the group consisting of myeloproliferative neoplasia (MPN), MPO-positive acute leukemia (preferably acute myeloid leukemia (AML)), MPO-positive chronic leukemia (preferably chronic myeloid leukemia (CML)), MPO-positive acute lymphoblastic leukemia (ALL), myelodysplastic syndrome and myeloproliferative syndrome, even more preferably acute myeloid leukemia (AML).

Preferably, the cells of said hematological neoplasia express myeloperoxidase on the protein level, more preferably on the mRNA level and the protein level. Preferably, leukemia cells of said leukemia express myeloperoxidase on the protein level, more preferably on the mRNA level and the protein level. Preferably, cells of said myeloproliferative neoplasia (MPN), acute leukemia, acute myeloid leukemia (AML), chronic leukemia, chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome and myeloproliferative syndrome express myeloperoxidase on the protein level, more preferably on the mRNA level and the protein level.

Preferably, said detection, diagnosis or prognosis is performed in vitro.

With respect to the different aspects of the present invention described above, in some embodiments the T cell receptor (or fragment thereof) according to any of the embodiments above, the soluble T cell receptor construct according to any of the embodiments above, the chimeric T cell receptor (or fragment thereof) according to any of the embodiments above, the bi-specific antibody according to any of the embodiments above, the nucleic acid or set of nucleic acids according to any of the embodiments above, the expression construct according to any of the embodiments above, the cell according to any of the embodiments above or the pharmaceutical composition as defined above is for use in the prevention and/or treatment of an MPO-positive hematological neoplasia, preferably of MPO-positive leukemia, more preferably of a disease selected from the group consisting of myeloproliferative neoplasia (MPN), MPO- positive acute leukemia (preferably acute myeloid leukemia (AML)), MPO-positive chronic leukemia (preferably chronic myeloid leukemia (CML)), MPO-positive acute lymphoblastic leukemia (ALL), myelodysplastic syndrome and myeloproliferative syndrome, even more preferably of acute myeloid leukemia (AML). Preferably, leukemia cells of said leukemia express myeloperoxidase on the mRNA and the protein level.

Preferably, said treatment involves administration of said T cell receptor (or fragment thereof), soluble T cell receptor construct, chimeric T cell receptor (or fragment thereof), bi-specific antibody, nucleic acid or set of nucleic acids, expression construct, cell or pharmaceutical composition to a patient in need thereof. Preferably, said patient is undergoing or has undergone treatment by chemotherapy and/or radiotherapy in combination with allogeneic stem cell transplantation (SCT) or in combination with autologous stem cell transplantation followed by deletion of transgenic T cells. Preferably, said patient is a human patient. Preferably, said patient is HLA-B *07:02 positive and/or HLA-B *07:04 positive and/or ULA-B*42:01 positive, more preferably HLA-B*07:02 positive. Preferably, said patient is having a relapse after treatment of said MPO-positive hematological neoplasia.

Preferably, said T cell receptor (or fragment thereof), soluble T cell receptor construct, chimeric T cell receptor (or fragment thereof), bi-specific antibody, nucleic acid or set of nucleic acids, expression construct, cell or pharmaceutical composition is configured for administration to a patient in need thereof. Preferably, said patient is undergoing or has undergone treatment by chemotherapy and/or radiotherapy in combination with allogeneic stem cell transplantation (SCT) or in combination with autologous stem cell transplantation followed by deletion of transgenic T cells. Preferably, said patient is a human patient. Preferably, said patient is HLA- B*07:02 positive and/or HLA-B *07:04 positive and/or HLA-B *42:01 positive, more preferably HLA-5 *07: 02 positive. Preferably, said patient is having a relapse after treatment of said MPO- positive hematological neoplasia.

Preferably, said treatment involves allogeneic stem cell transplantation (SCT) from a donor to said patient.

Preferably, said treatment also involves a combination of chemotherapy and/or radiotherapy with allogeneic stem cell transplantation (SCT) from a donor to said patient. Preferably, said treatment involves administration of a donor lymphocyte infusion to said patient, wherein, preferably, the lymphocytes of said donor lymphocyte infusion are transgenic T cells expressing said T cell receptor, more preferably transgenic CD3 ⁺ or CD8 ⁺ T cells expressing said T cell receptor.

Preferably, said patient is HLA-B *07: 02 positive and/or HLA-B*07:04 positive and/or HLA- B*42:01 positive, more preferably HLA-B*07:02 positive, whereas said donor is HLA-B*07:02 negative and/or BLA-B*07:04 negative and/or HLA-B*42:01 negative, more preferably HLA- B*07:02 negative. Alternatively, said patient and said donor are both HLA-B *07: 02 positive and/or HLA-B *07:04 positive and/or HLA-B *42:07 positive, more preferably HLA-B*07:02 positive, whereas this will necessitate an efficient suicide mechanism to eliminate the MPO- specific cell/substance.

Preferably said allogeneic stem cell transplantation is a haploidentical or one allele mismatch allogeneic stem cell transplantation, wherein, preferably, said patient is HLA-B *07/ 02 positive and/or HLA-B *07/04 positive and/or HLA-B *42; 01 positive, more preferably HLA-B*07:02 positive, and said donor is HLA-B *07:02 negative and/or HLA-B *07:0¥ negative and/or HLA- B*42:01 negative, more preferably HLA-j3*07:02 negative. Alternatively, said allogeneic stem cell transplantation is an HLA-identical allogeneic stem cell transplantation, wherein, preferably, said patient and said donor are both HLA-B *07:02 positive and/or HLA-B *07:04 positive and/or HLA-B *42: 01 positive, more preferably HLA-B*07:02 positive.

As the skilled person will appreciate, if said patient and said donor are HLA-identical, then the transgenic T cells expressing said T cell receptor must include a mechanism that allows to reduce the number of transgenic T cells in said patient after treatment, in order to allow for regeneration of healthy (i.e. non-leukemic) myeloid cells/myeloid precursor cells in the bone marrow. This can for example be achieved by a "suicide mechanism" as described in Bonini et al., 1997.

Preferably, said treatment comprises the use of adoptive, target cell specific immunotherapy or genetic immunization. In a tenth aspect, the present invention relates to the use of the T cell receptor (or fragment thereof) according to any of the embodiments above, the soluble T cell receptor construct according to any of the embodiments above, the chimeric T cell receptor (or fragment thereof) according to any of the embodiments above, the bi-specific antibody according to any of the embodiments above, the nucleic acid or set of nucleic acids according to any of the embodiments above, the expression construct according to any of the embodiments above, the cell according to any of the embodiments above or the pharmaceutical composition as defined above for the manufacture of a medicament, preferably a medicament for the prevention and/or treatment of an MPO-positive hematological neoplasia, preferably of MPO-positive leukemia, more preferably of a disease selected from the group consisting of myeloproliferative neoplasia (MPN), MPO- positive acute leukemia (preferably acute myeloid leukemia (AML)), MPO-positive chronic leukemia (preferably chronic myeloid leukemia (CML)), MPO-positive acute lymphoblastic leukemia (ALL), myelodysplasia syndrome and myeloproliferative syndrome, even more preferably of acute myeloid leukemia (AML).

In an eleventh aspect, the present invention relates to a method of preventing and/or treating an MPO-positive hematological neoplasia, preferably MPO-positive leukemia, more preferably a disease selected from the group consisting of myeloproliferative neoplasia (MPN), MPO-positive acute leukemia (preferably acute myeloid leukemia (AML)), MPO-positive chronic leukemia (preferably chronic myeloid leukemia (CML)), MPO-positive acute lymphoblastic leukemia (ALL), myelodysplastic syndrome and myeloproliferative syndrome, even more preferably acute myeloid leukemia (AML),

comprising the steps of

(a) providing lymphocytes of a patient or a (stem cell) donor;

(b) providing one or more of

(i) a T cell receptor (or fragment thereof) according to any of the embodiments above or a chimeric T cell receptor according to any of the embodiments above,

(ii) a nucleic acid or set of nucleic acids according to any of the embodiments above,

(iii) an expression construct according to any of the embodiments above,

(c) ex vivo introduction of one or more of (i) to (iii) of step (b) into the lymphocytes of step (a) and, thereby, obtaining genetically modified lymphocytes, (d) administering the genetically modified lymphocytes of step (c) to a subject or patient in need thereof;

(e) optionally, combining any of the steps (b) to (d) with radiation, checkpoint inhibitor or cancer chemotherapy.

In such method, said MPO-positive hematological neoplasia, said lymphocytes, said patient, said donor and said genetically modified lymphocytes are preferably as defined in the above- described aspects of the present invention or any of the embodiments referring to them.

T cells express inhibitory receptors such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) or programmed cell death 1 (PD-1), especially after long term exposure to the antigen. The ligands of CTLA-4 and PD-1, B7-1/B7-2 and PD-L1/PD-L2 respectively, as well as the ligand of ΤΓΜ-3, Galectin 9, are frequently expressed on tumor tissue and are involved in induction of T cell tolerance and thus are called immune checkpoints. To avoid silencing of transferred myeloperoxidase specific T cells checkpoint inhibitors or immune checkpoint inhibitors, exemplified by but not restricted to anti-CTLA-4, anti-PD-Ll or anti-PD-1 antibodies, can be used to avoid inhibition of T cells within the tumor microenvironment (described in Hamid et al. (2013)).

The subjects or patients are preferably HLA-B*07:02 positive and/or HLA-B*07:04 positive and/or BLA-B*42:01 positive, more preferably HLA-B*07:02 positive.

Preferably, the lymphocytes provided in step (a) are T cells, NK cells and/or NKT cells, more preferably T cells, even more preferably CD3 ⁺ T cells or CD8 ⁺ T cells.

The lymphocytes provided in step (a) can be obtained from the subject or patient, such as from the blood or by leukapheresis of allogeneic stem cell donors.

Preferably, the ex vivo introduction in step (c) is carried out via electroporation of a nucleic acid of the present invention or of an expression construct of the present invention, or by transfection reagents, such as liposomes, or by transient RNA transfection. The expression construct is preferably, as described above, a viral construct, preferably a lentiviral vector, retroviral vector, or other state of the art viral vector.

Examples for suitable expression constructs are viral vectors, such as

- retroviral vectors, such as MP71 vectors (as described in Engels et al., 2003), or retroviral SIN vectors (as described in Yu et al., 1986);

and

- lentiviral vectors or lentiviral SIN vectors, as described in Miyoshi et al., 1998.

Another example for a suitable expression construct is the Sleeping Beauty (SB) transposon transposase DNA plasmid system, SB DNA plasmid (as described in Maiti et al., 2013). Alternatively, the vector is a vector suitable for the piggyBac (as described in Manuri PV et al., 2010) transposition system or a vector for chromosomal insertion through the CRISPR/CAS9 system (Mandal PK et al., 2014).

In a twelfth aspect, the present invention relates to a method of preventing and/or treating an MPO-positive hematological neoplasia, preferably MPO-positive leukemia, more preferably a disease selected from the group consisting of myeloproliferative neoplasia (MPN), MPO-positive acute leukemia (preferably acute myeloid leukemia (AML)), MPO-positive chronic leukemia (preferably chronic myeloid leukemia (CML)), MPO-positive acute lymphoblastic leukemia (ALL), myelodysplastic syndrome and myeloproliferative syndrome, even more preferably acute myeloid leukemia (AML),

comprising the steps of

(a) providing one or more of

(i) a T cell receptor (or fragment thereof) according to any of the embodiments above or a soluble T cell receptor construct according to any of the embodiments above or a chimeric T cell receptor (or fragment thereof) according to any of the embodiments above or a bi-specific antibody according to any of the embodiments above,

(ii) a nucleic acid or set of nucleic acids according to any of the embodiments above, (iii) an expression construct according to any of the embodiments above,

(iv) a cell according to any of the embodiments above, and

(v) a pharmaceutical composition as defined above;

(b) direct application, preferably via injection or infusion, of one or more of (i) to (v) of step (a) to a subject or patient in need thereof,

(c) optionally, combining step (b) with radiation, checkpoint inhibitor or cancer chemotherapy.

In such method, said MPO-positive hematological neoplasia, said patient and said expression construct are preferably as defined in the above-described aspects of the present invention or any of the embodiments referring to them.

Preferably, the bi-specific antibodies or the soluble TCR constructs of the present invention are used as recombinant proteins.

In some embodiments, in step (b) the one or more of (i) to (v) of step (a) are used as recombinant proteins.

The recombinant proteins, preferably the bi-specific antibodies or the soluble TCR constructs of the present invention, are expressed, preferably in E. coli or mammalian cells, and then purified before the application.

In a thirteenth aspect, the present invention relates to a method of detecting, diagnosing, prognosing, preventing and/or treating a hematological neoplasia, preferably leukemia, more preferably a disease selected from the group consisting of myeloproliferative neoplasia (MPN), acute leukemia (preferably acute myeloid leukemia (AML)), chronic leukemia (preferably chronic myeloid leukemia (CML)), acute lymphoblastic leukemia (ALL), myelodysplasia syndrome and myeloproliferative syndrome, even more preferably acute myeloid leukemia (AML),

comprising the detection and/or destruction of (leukemic) cancer cells of a patient with the use of the soluble T cell receptor construct according to any of the embodiments above. Preferably, said cancer cells express myeloperoxidase at the protein level, more preferably at the mRNA level and at the protein level.

Preferably said cancer cells are MPO-positive hematological neoplasia cells, more preferably MPO-positive leukemia, even more preferably cancer cells selected from the group consisting of myeloproliferative neoplasia (MPN) cells, MPO-positive acute leukemia cells (preferably acute myeloid leukemia (AML) cells), MPO-positive chronic leukemia cells (preferably chronic myeloid leukemia (CML) cells), MPO-positive acute lymphoblastic leukemia (ALL) cells, myelodysplasia syndrome cells and myeloproliferative syndrome cells. Even more preferably, said cancer cells are acute myeloid leukemia (AML) cells.

In such method, said MPO-positive hematological neoplasia cells and said patient are preferably as defined in the above-described aspects of the present invention or any of the embodiments referring to them.

Soluble T cell receptor constructs can be applied to patients:

- by intravenous infusions,

- by subcutaneous injection or infusion,

- by intramuscular injection,

- by inhalation or by spraying to the mucosal surfaces of the respiratory tract.

Suitable dose ranges for application of soluble T cell receptor constructs vary between 1 mg to 10 000 mg dependent on the application method and application site. Soluble T cell receptors can bind to cells presenting myeloperoxidase peptides on their HLA-B*07:02/B*07:04/B*42:01 molecules, namely MPO-positive hematological neoplasia cells, such as more preferably MPO- positive leulcemia, for example myeloproliferative neoplasia (MPN) cells, MPO-positive acute leukemia cells (e.g. acute myeloid leukemia (AML) cells), MPO-positive chronic leukemia cells (e.g. chronic myeloid leukemia (CML) cells), MPO-positive acute lymphoblastic leukemia (ALL) cells, myelodysplasia syndrome cells or myeloproliferative syndrome cells, in which a peptide of myeloperoxidase residues 466-474 is presented by HLA-B*07:02/B*07:04/B*42:01. Soluble T cell receptors can induce destruction of the cells via different methods, such as radiation by linked radionuclides or killing by toxins such as pseudomonas exotoxin. In addition, soluble T cell receptors with linked Fc-domains can redirect and activate NK-cells and/or monocytes for the lysis of cells of a MPO-positive hematological neoplasia, in particular cells of an MPO-positive leukemia as described above. Similarly, soluble T cell receptors linked to anti- CD3 -antibodies can redirect and activate T cells for lysis of cells of a MPO-positive hematological neoplasia, in particular cells of an MPO-positive leukemia as described above. Furthermore, soluble T cell receptors combined with a linker can redirect and active T cells that are transgenic for a CAR that binds specifically to the linker.

In a fourteenth aspect, the present invention relates to a method of detecting a hematological neoplasia, preferably leukemia, more preferably a disease selected from the group consisting of myeloproliferative neoplasia (MPN), acute leukemia (preferably acute myeloid leukemia (AML)), chronic leukemia (preferably chronic myeloid leukemia (CML)), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome and myeloproliferative syndrome, even more preferably acute myeloid leukemia (AML),

comprising the in vitro staining of cells obtained from a human subject by using the soluble T cell receptor construct of the present invention which is labelled.

Preferably, the cells of said hematological neoplasia express myeloperoxidase on the protein level, more preferably on the mRNA level and the protein level. Preferably, leukemia cells of said leukemia express myeloperoxidase on the protein level, more preferably on the mRNA level and the protein level. Preferably, cells of said myeloproliferative neoplasia (MPN), acute leukemia, acute myeloid leukemia (AML), chronic leukemia, chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome and myeloproliferative syndrome express myeloperoxidase on the protein level, more preferably on the mRNA level and the protein level.

In such method, said hematological neoplasia is preferably as defined in the above-described aspects of the present invention or any of the embodiments referring to them. Preferably, said human subject is HLA~B*07:02 positive and/or HLA-B*07:04 positive and/or HLA-B*42:01 positive, more preferably HLA-B*07:02 positive.

Preferably, said cells obtained from said human subject are bone marrow aspirate cells or peripheral blood mononucleated cells cells. Preferably, said cells obtained from said human subject are cells isolated from blood or bone marrow or chloroma/myeloid sarcoma cells (leukemic manifestations outside from blood/bone marrow indicates an extramedullary myeloid tumor).

The label can be fluorophore(s), gold and/or radionuclide(s).

Soluble T cell receptors linked to fluorescent molecules such as fluorescein can be used to stain cells of a MPO-positive hematological neoplasia, in particular cells of an MPO-positive leukemia as described above in vitro. For this purpose, said cells , are incubated in vitro with the soluble T cell receptor constructs, and cells expressing HLA-5*07:02/myeloperoxidase peptide complexes, HLA-5*07:0¥/myeloperoxidase peptide complexes and/or HLA- 5*¥2/0i/myeloperoxidase peptide complexes on the cell surface can be detected according to their fluorescence by a Fluorescence activated cell sorter (FACS) or by microscope.

Alternatively, labeled soluble T cell receptor constructs (e.g. with fluorochromes, gold or radionuclides) are injected into a subject or patient and allow detection and, if the soluble T cell receptor carries an appropriate label, subsequent destruction of myeloperoxidase-positive tumor, tumor metastasis or minimal residual disease after tumor resection.

The soluble TCR constructs according to the invention are suitable to be directly used for the detection and destruction of cells of a MPO-positive hematological neoplasia, in particular cells of an MPO-positive leukemia as described above, without the need of T cells.

The term "T cell receptor", as used in the present invention, has the common meaning. Thus, a T cell receptor is a protein receptor that is capable of recognizing antigenic determinants (peptides) presented in the MHC (major histocompatibility complex) context. Typically, a T cell receptor is composed of a heterodimer of a T cell receptor alpha (a) chain and a T cell receptor beta (β) chain. For more details, see Murphy, Travers, Walport: "Janeway's Immunobiology", 7th ed., Garland Science (2008).

The T cell receptor of the HLA-class I-restricted CD8+ CTL consists of two proteins, the TCR ct- chain and the TCR β-chain. Both the TCR α-chain and the TCR β-chain are formed from various gene segments, which are rearranged from a variety of variable gene segments at the TCR a locus and the TCR β locus. The TCR a-chain consists of a variable (V) gene segment, a J (Joining) gene segment and a constant (C) gene segment. The TCR β-chain consists of a variable (V) gene segment, a J (Joining) gene segment, a D (diversity) gene segment and a constant (C) gene segment.

The individual TCR of individual T cells is generated during the thymocyte development by recombination of gene segments. D-to-J recombination occurs first in the β chain of the TCR. This process can involve either the joining of the ϋβΐ gene segment to one of six jpi segments or the joining of the ϋβ2 gene segment to one of seven Jβ2 segments. DJ recombination is followed (as above) with Vβ-to-DβJβ rearrangements. All gene segments between the νβ-ϋβ^β gene segments in the newly formed complex are deleted and the primary transcript is synthesized that incorporates the constant domain gene (νβ-Όβ^β^β). mRNA transcription splices out any intervening sequence and allows translation of the full length protein for the TCR Cβ chain.

The rearrangement of the alpha (a) chain of the TCR follows β chain rearrangement, and resembles V-to-J rearrangement described for Ig light chains (see above). The diversity of the somatically rearranged TCRs is enhanced further by the introduction of additional nucleotides between the V and J gene segments of rearranged TCR-alpha genes. The rearrangement of TCR gene segments provide a diverse repertoire of approximately 10 ¹⁸ different TCRs. The TCR- alpha-chain and the TCR-beta-chain pair after their synthesis to yield the αβ-TCR -heterodimer that is expressed on a majority of T cells.

The specificity of the recognition of viral or tumor-associated or autoimmunity-associated peptides is mainly determined by the CDR3 -region of both the TCR-alpha and the TCR-beta chains. Each CTL usually expresses only a single functional T cell receptor (TCR) that allows the recognition of only a single antigenic peptide (or group of peptides with highly related sequence). Dependent on the individual TCR sequence, TCRs can exhibit a certain cross- reactivity that may allow also the recognition of peptide variants to a certain degree.

At some instances, the present invention indicates that a certain amino acid sequence A has "at least x % identity" with another amino acid sequence B [or to a certain nucleotide sequence A that has "at least x % identity" with another nucleotide sequence B]. This designates a situation where, in an optimal sequence alignment of sequence A with sequence B at least x % of the aligned amino acid residues [or nucleotides] of sequence A and B are identical. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software, and those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Preferably, the alignment is carried out according to the algorithm of Huang and Miller (published in Adv. Appl. Math. (1991) 12:337-357), for example as global alignment with the software ALIGN (by William Pearson, available at http://www.ch.embnet.org/ software/L ALIGN_form.html), with an opening gap penalty setting of -14, an extending gap penalty setting of -4, and with the scoring matrix BLOSUM80. The amino acid residues [or nucleotides] at corresponding amino acid [or nucleotide] positions are then compared. When a position in the first sequence is occupied by the same amino acid residue [or nucleotide] as the corresponding position in the second sequence, then the molecules are identical at that position.

At some instances, the present application refers to a "conservative amino acid exchange", for example in the context that all amino acid exchanges by which a certain first amino acid sequence differs from a certain second are "conservative amino acid exchanges". This is meant to designate that corresponding, but differing residue positions in the two amino acid sequences are occupied by amino acids that have residues with similar side chains. Groups of amino acids that have similar side chains are: For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic- hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids exchange groups are: valine- leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.

The terms "HLA-B*07;02" and "HLA-B7" are used interchangeably herein and refer to a certain HLA allele and the corresponding protein that is capable of binding to the peptide MPO ₄ 6 _6-474 .

If the present application indicates that a certain molecule or part of a molecule (such as the T cell receptor α-chain or the constant region of the T cell receptor α-chain) "is composed of human sequences", this designates a situation where said molecule or part of a molecule comprises only sequences of human origin, but no sequences originating from other organisms. Similarly, if the present application indicates that a certain molecule or part of a molecule "is composed of mouse sequences", this designates a situation where said molecule or part of a molecule comprises only sequences that originate from mouse, but no sequences originating from other organisms. If the present application indicates that a certain molecule or part of a molecule "is composed of a combination of human and mouse sequences", this designates a situation where said molecule or part of a molecule comprises sequence(s) originating from mouse and sequence(s) of human origin, but no sequences originating from other organisms.

The terms "MPO ₄ 6 _6-474 " and "MPO ₅ " are used interchangeably herein and refer to a peptide corresponding to residues 466-474 of the protein human myeloperoxidase, i.e. a peptide with the sequence of SEQ ID NO: 7 (NPRWDGERL).

At some instances, the present application refers to an "MPO-positiye" disease, for example "MPO-positive leukemia". This means that the cells of said leukemia express myeloperoxidase both at the rnRNA level and at the protein level. Similarly, if the present application refers to certain "MPO-positive" cells, for example "MPO-positive leukemia cells", this means that said cells express myeloperoxidase both at the mRNA level and at the protein level. "Hematological neoplasia", as used herein, refers to a malignant disease of the hematopoietic system involving cells of the myeloid and/or lymphoid lineage characterized by the progressive proliferation of malignant cells of said lineages.

"Leukemia" as used herein, refers to a disease involving the progressive proliferation of abnormal leukocytes found in hematopoietic tissues, other organs and in the blood, resulting in increased numbers of leukocytes. "Leukemic cells" refers to leukocytes characterized by an increased abnormal proliferation of cells. Leukemic cells may be obtained from a subject diagnosed with leukemia. The term includes, but is not limited to, acute lymphocytic leukemia (ALL), myeloproliferative neoplasias (MPN), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), myelodysplastic syndrome, myeloproliferative syndrome, hypereosinophilic syndrome (such as myeloid neoplasia with eosinophilia or chronic eosinophilic leukemia), extramedullary myeloid tumors (such as chloromas or myeloid sarcomas) and monocytic leukemia. In some embodiments, the term "leukemia" is limited to leukemias that express myeloperoxidase at the protein level, preferably at the mRNA level and at the protein level, and the term "leukemic cells" is limited to leukemic cells that express myeloperoxidase at the protein level, preferably at the mRNA level and at the protein level.

The term "treatment", as used herein, refers to the process of providing a subject with a pharmaceutical treatment, such that a disease or disease state is alleviated, reduced, minimized, halted or even healed, and/or such that the chances of a relapse into the disease state are reduced or a relapse into the disease state is even prevented.

"Pharmaceutically acceptable", as used herein for example in the context of a pharmaceutically acceptable salt of a compound, refers to a substance (or composition) that is non-toxic to the subject to which it is administered and that thus can be used in the formulation of a pharmaceutical product. If the pharmaceutically acceptable substance is part of a pharmaceutical composition, then the term also implies that the pharmaceutically acceptable substance is compatible with the other ingredients of the said pharmaceutical composition.

"Cultured cells", as used herein, refers to cells growing under conditions of in vitro culture. Thus cultured cells are free tissue cells that are cultivated in a system entirely apart from their normal environment in which the conditions and factors for growth can be varied at will within the boundaries tolerated by the cells.

As used herein, the term "in vitro" means occurring outside of a living organism. The term in vitro can describe processes/conditions occurring within a cell culture system. In contrast to "in vitro", the term "in vivo" means occurring within a living organism. The term "ex vivo" means occurring in a cell culture context or tissue culture context. For example, cells may be extracted from a subject; a therapeutic gene may be introduced into the cells in a cell culture context outside of the living organism (i.e. ex vivo); and subsequently the transgenic cells may be returned into the same subject.

SEQ ID NO: 1 shows the amino acid sequence of the CDRl (Complementarity Determining Region 1) region of the α-chain of a T cell receptor according to the present invention (CDRl of T cell receptor a-chain):

YGATPY

SEQ ID NO: 2 shows the amino acid sequence of the CDR2 (Complementarity Determining Region 2) region of the α-chain of a T cell receptor according to the present invention (CDR2 of T cell receptor a-chain):

YFSGDTLV

SEQ ED NO: 3 shows the amino acid sequence of the CDR3 (Complementarity Determining Region 3) region of the α-chain of a T cell receptor according to the present invention (CDR3 of T cell receptor a-chain):

CAGRAAGNKLTF

SEQ ID NO: 4 shows the amino acid sequence of the CDRl (Complementarity Determining Region 1) region of the β-chain of a T cell receptor according to the present invention (CDRl of T cell receptor β-chain):

MNHEY SEQ ID NO: 5 shows the amino acid sequence of the CDR2 (Complementarity Determining Region 2) region of the β-chain of a T cell receptor according to the present invention (CDR2 of T cell receptor β-chain):

SVGEGT

SEQ ID NO: 6 shows the amino acid sequence of the CDR3 (Complementarity Determining Region 3) region of the β-chain of a T cell receptor according to the present invention (CDR3 of T cell receptor β-chain):

CASSYSSGQPQHF

SEQ ID NO: 7 shows the amino acid sequence of myeloperoxidase, residues 466 to 474

(MPO ₄ 66-474):

NPRWDGERL

SEQ ID NO: 8 shows the amino acid sequence of the α-chain of a T cell receptor according to the present invention (TCR2.5D6), wherein said α-chain is composed of a combination of the variable region of a human α-chain (variable region in SEQ ID NO: 8: amino acids 1 to 130 of SEQ ID NO: 8) and the constant region of a mouse α-chain and wherein, in the constant region of the mouse α-chain, an amino acid exchange T83C has been carried out (Voss et al., 2010): LLELIPLLGIHFVLRTARAQSVTOPDIHITVSEGASLELRCNYSYGATPYLFWYVOSPGO G LOLLLKYFSGDTLVQGIKGFEAEFKRSQSSFNLRKPSVHWSDAAEYFCAGRAAGNKLTF GGGTRVLVKPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVL DMKAMDSKSNGAIAWSNQTSFTCQDIFKETNACYPSSDVPCDATLTEKSFETDMNLNF QNLS VMGLRILLLKVAGFNLLMTLRL WS S

In some embodiments, the amino acid sequence of a full-length T cell receptor α-chain according to the present invention may comprise an additional N-terminal M (methionine) residue.

SEQ ID NO: 9 shows the amino acid sequence of the β-chain of a T cell receptor according to the present invention (TCR2.5D6), wherein said β-chain is composed of a combination of the variable region of a human β-chain (variable region in SEQ ID NO: 9: amino acids 1 to 130 of SEQ ID NO: 9) and the constant region of a mouse β-chain and wherein, in the constant region of the mouse β-chain, an amino acid exchange S79C has been carried out (Voss et al., 2010):

SLGLLCCGVFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYROD

PGMGLRLIHYSVGEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSOTSVYFCASSY SS

G^P^HFGDGTPXSILEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDIIVELS W

WVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVCATFWHNPRNHFRCQVQFHGLSEE D

KWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVS GL

VLMAMVKKKNS

In some embodiments, the amino acid sequence of a full-length T cell receptor β-chain according to the present invention may comprise an additional N-terminal M (methionine) residue.

SEQ ID NO: 10 shows a DNA sequence of the α-chain of a T cell receptor according to the present invention (TCR2.5D6; CDRs indicated by underline):

CTGCTGGAACTGATCCCCCTGCTGGGAATCCACTTCGTGCTGAGGACAGCCAGAGCC

CAGTCTGTGACCCAGCCCGACATCCACATCACCGTGTCTGAGGGCGCCAGCCTGGA

ACTGCGGTGCAACTACTCTTACGGCGCCACCCCCTACCTGTTTTGGTACGTGCAGAG

CCCCGGACAGGGACTGCAGCTGCTGCTGAAGTACTTTAGCGGCGACACCCTGGTGC

AGGGCATCAAGGGATTCGAGGCCGAGTTCAAGCGGAGCCAGAGCAGCTTCAACCTG

CGGAAGCCCTCCGTGCATTGGAGCGACGCCGCCGAGTATTTCTGTGCCGGCAGAGC

AGCCGGCAACAAGCTGACATTTGGCGGCGGAACCAGGGTGCTCGTGAAGCCCAACA

TCCAGAACCCCGAGCCCGCCGTGTACCAGCTGAAGGACCCTAGAAGCCAGGACAGC

ACCCTGTGCCTGTTCACCGACTTCGACTCCCAGATCAACGTGCCCAAGACCATGGAA

AGCGGCACCTTCATCACCGACAAGACCGTGCTGGACATGAAGGCCATGGACAGCAA

GAGCAACGGCGCCATTGCCTGGTCCAATCAGACCAGCTTCACATGCCAGGACATCTT

CAAAGAGACAAACGCCTGCTACCCCAGCAGCGACGTGCCCTGTGATGCCACCCTGA

CCGAGAAGTCCTTCGAGACAGACATGAATCTGAACTTCCAGAACCTGAGCGTGATG

GGCCTGAGAATCCTGCTGCTGAAAGTGGCCGGCTTCAATCTGCTGATGACCCTGAGA

CTGTGGTCCAGC

SEQ ID NO: 11 shows a DNA sequence of the a-chain of a T cell receptor according to the present invention (TCR2.5D6) including start and stop codons (CDRs indicated by underline): ATGCTGCTGGAACTGATCCCCCTGCTGGGAATCCACTTCGTGCTGAGGACAGCCAGA

GCCCAGTCTGTGACCCAGCCCGACATCCACATCACCGTGTCTGAGGGCGCCAGCCTG

GAACTGCGGTGCAACTACTCTTACGGCGCCACCCCCTACCTGTTTTGGTACGTGCAG

AGCCCCGGACAGGGACTGCAGCTGCTGCTGAAGTACTTTAGCGGCGACACCCTGGT

GCAGGGCATCAAGGGATTCGAGGCCGAGTTCAAGCGGAGCCAGAGCAGCTTCAACC

TGCGGAAGCCCTCCGTGCATTGGAGCGACGCCGCCGAGTATTTCTGTGCCGGCAGA

GCAGCCGGCAACAAGCTGACATTTGGCGGCGGAACCAGGGTGCTCGTGAAGCCCAA

CATCCAGAACCCCGAGCCCGCCGTGTACCAGCTGAAGGACCCTAGAAGCCAGGACA

GCACCCTGTGCCTGTTCACCGACTTCGACTCCCAGATCAACGTGCCCAAGACCATGG

AAAGCGGCACCTTCATCACCGACAAGACCGTGCTGGACATGAAGGCCATGGACAGC

AAGAGCAACGGCGCCATTGCCTGGTCCAATCAGACCAGCTTCACATGCCAGGACAT

CTTCAAAGAGACAAACGCCTGCTACCCCAGCAGCGACGTGCCCTGTGATGCCACCCT

GACCGAGAAGTCCTTCGAGACAGACATGAATCTGAACTTCCAGAACCTGAGCGTGA

TGGGCCTGAGAATCCTGCTGCTGAAAGTGGCCGGCTTCAATCTGCTGATGACCCTGA

GACTGTGGTCCAGCTGA

SEQ ID NO: 12 shows a DNA sequence of the β-chain of a T cell receptor according to the present invention (TCR2.5D6; CDRs indicated by underline):

TCTCTGGGACTGCTGTGTTGCGGCGTGTTCAGCCTGCTGTGGGCCGGACCTGTGAAT

GCCGGCGTGACCCAGACCCCCAAGTTCCGGGTGCTGAAAACCGGCCAGAGCATGAC

CCTGCTGTGCGCCCAGGACATGAACCACGAGTACATGTATTGGTACAGACAGGACC

CCGGCATGGGCCTGCGGCTGATCCACTATTCTGTGGGCGAGGGCACCACCGCCAAG

GGCGAAGTGCCTGATGGCTACAACGTGTCCCGGCTGAAGAAGCAGAACTTCCTGCT

GGGCCTGGAAAGCGCCGCTCCTAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCT

ACAGCAGCGGCCAGCCTCAGCACTTTGGCGACGGCACCAGACTGAGCATCCTGGAA

GATCTGCGGAACGTGACCCCCCCCAAGGTGTCCCTGTTCGAGCCTAGCAAGGCCGA

GATCGCCAACAAGCAGAAAGCCACCCTCGTGTGCCTGGCCAGAGGCTTCTTCCCCG

ACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTGTCC

ACCGATCCCCAGGCCTACAAAGAGAGCAACTACAGCTACTGCCTGTCCAGCAGACT

GAGAGTGTGCGCCACCTTCTGGCACAACCCCCGGAACCACTTCAGATGCCAGGTGC

AGTTTCACGGCCTGAGCGAAGAGGACAAGTGGCCCGAGGGCAGCCCTAAGCCCGTG

ACACAGAATATCAGCGCCGAAGCCTGGGGCAGAGCCGACTGTGGAATCACCAGCGC CAGCTACCATCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATTCTGCTGGGCAA GGCCACCCTGTACGCCGTGCTGGTGTCTGGCCTGGTGCTGATGGCCATGGTCAAGAA GAAGAACAGC

SEQ ID NO: 13 shows a DNA sequence of the β-chain of a T cell receptor according to the present invention (TCR2.5D6) including start and stop codons (CDRs indicated by underline):

ATGTCTCTGGGACTGCTGTGTTGCGGCGTGTTCAGCCTGCTGTGGGCCGGACCTGTG

AATGCCGGCGTGACCCAGACCCCCAAGTTCCGGGTGCTGAAAACCGGCCAGAGCAT

GACCCTGCTGTGCGCCCAGGACATGAACCACGAGTACATGTATTGGTACAGACAGG

ACCCCGGCATGGGCCTGCGGCTGATCCACTATTCTGTGGGCGAGGGCACCACCGCC

AAGGGCGAAGTGCCTGATGGCTACAACGTGTCCCGGCTGAAGAAGCAGAACTTCCT

GCTGGGCCTGGAAAGCGCCGCTCCTAGCCAGACCAGCGTGTACTTCTGCGCCAGCA

GCTACAGCAGCGGCCAGCCTCAGCACTTTGGCGACGGCACCAGACTGAGCATCCTG

GAAGATCTGCGGAACGTGACCCCCCCCAAGGTGTCCCTGTTCGAGCCTAGCAAGGC

CGAGATCGCCAACAAGCAGAAAGCCACCCTCGTGTGCCTGGCCAGAGGCTTCTTCC

CCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTG

TCCACCGATCCCCAGGCCTACAAAGAGAGCAACTACAGCTACTGCCTGTCCAGCAG

ACTGAGAGTGTGCGCCACCTTCTGGCACAACCCCCGGAACCACTTCAGATGCCAGGT

GCAGTTTCACGGCCTGAGCGAAGAGGACAAGTGGCCCGAGGGCAGCCCTAAGCCCG

TGACACAGAATATCAGCGCCGAAGCCTGGGGCAGAGCCGACTGTGGAATCACCAGC

GCCAGCTACCATCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATTCTGCTGGGC

AAGGCCACCCTGTACGCCGTGCTGGTGTCTGGCCTGGTGCTGATGGCCATGGTCAAG

AAGAAGAACAGCTGA

In connection with their finding that myeloperoxidase (MPO) is an essential and distinct marker for myeloid cells and AML that is restrictedly expressed in the hematopoietic system (Klar et al., 2014), the inventors have identified a T cell receptor from the naive T cell repertoire of an HLA- B7-negative (i.e. HLA-B*07:02 negative) donor with specificity against one selected HLA- B7/B42:01 -restricted MPO epitope (MPO5). This T cell receptor shows high reactivity against leukemia cells endogenously expressing myeloperoxidase (MPO), and T cells transgenic for this TCR recognize MPO ⁺ leukemic cell lines and MPO ⁺ primary leukemic cells from patients with AML and MPN in vitro and in vivo. On target reactivity was limited to hematopoietic cells with high MPO-mRNA expression. MPO-dependent toxicity was not observed against mature hematopoietic cells with MPO protein but low mRNA expression, and no reactivity against monocytes and healthy hematopoietic stem cells (HSC) and only very low reactivity against granulocytes were observed. Moreover, no off-target reactivity was observed when diverse cell lines of different origin as well as a high number of peptides that have been eluted from HLA-B7 were tested. Furthermore, the peptide motif recognized by TCR-transgenic T cells is unique for MPO and reactivity against peptides with high homology to MPO5 is lacking. This implies that adoptive T cell therapy with T cells transgenic for this TCR may be safe in the haploidentical or HLA-B7 allele mismatch allogeneic SCT setting, where donor myeloid progenitor cells lack the HLA-B7 restricting allele and thus this approach can be the base for a personalized therapeutic option for patients with relapsing leukemias by adoptive T cell therapies using a defined tumor- specific T cell receptor.

The unique features of the TCR of the present invention, TCR2.5D6, are its strong anti-tumor- reactivity in vitro as well as in vivo, and its enormously high specificity. These features strongly depend on the combination of the specific sequences of the CDR3 regions as well as the CDR1 and CDR2 regions of the TCR2.5D6, which is specific for the donor from whose T cells TCR2.5D6 was isolated. Thus, the skilled artisan who might have information about the methodology for isolating TCRs, such as described in Klar et al., 2014, is not able to determine the specific sequence of TCR2.5D6.

In the following, reference is made to the figures:

All methods mentioned in the figure descriptions below were carried out as described in detail in the examples.

DRAWINGS Figure 1 shows experimental data confirming MPO as potential T cell target antigen in myeloid leukemia.

(A) MPO-derived HLA ligands identified by the immunopeptidomic approach were synthesized, sequenced by mass spectrometry and matched to the spectra of eluted peptides after normalization with the mMass tool for sequence validation. Spectra of synthetic peptides are shown in red (lower) and spectra of eluted peptides in black (upper).

(B) mRNA expression of MPO in relation to PBMC was analyzed by qPCR on primary material and diverse healthy tissues. Ct values were normalized against the geometric mean of three housekeeping genes (GAPDH, HPRT1, HMBS) using the ddCT method.

(C) MPO immunohistochemistry in diverse healthy tissue samples (from left to right and top to bottom): bone marrow, lymph node, spleen, lung, heart, kidney, brain, thyroid gland, endometrium, esophagus, intestinal mucosa, colon mucosa, liver, vessels, soft tissue.

Figure 2 shows experimental data on the expansion of T cells specific for the HLA-B7-restricted peptide MPO ₅ and the functionality of lymphocytes transgenic for an MPO ₅ -specific TCR.

(A) Phenotyping of single HLA-mismatched dendritic cells (DCs) for stimulation with MPO ₅ . Dendritic cells from an HLA-B*07:02 negative donor were matured and electroporated with HLA-5*07:02P2AeGFP encoding in vitro transcribed RNA. The degree of maturation of the DCs was determined through the surface markers CD83, CD80 and CD86. Successful electroporation was verified by measuring eGFP expression.

(B) MPO ₅ peptide-specific T cells were expanded after stimulation of naive T cells from an HLA-B7 negative donor with peptide-pulsed (0.1 μΜ) single HLA-mismatched DCs. Peptide specificity of expanded T cells was investigated with an MPO ₅ -specific HLA multimer by flow cytometry.

(C) Peptide-specific, lytic capacity of cloned T cells at estimated effector to target ratios (25:1) was tested by ⁵¹ Cr assay (the mean of duplicates is shown).

(D) Stimulated peripheral lymphocytes from a healthy donor were transduced with TCR2.5D6 and stained with anti-CD4, anti-CD8, the MPO ₅ specific HLA multimer and the TCRm antibody, which is directed against the murinized constant region of the optimized TCR. Binding of MPO ₅ specific HLA multimer and expression of TCRm is shown in total lymphocytes as well as T cell subpopulations positive for CD4 and CD 8. (E) For functional characterization, transduced lymphocytes were co-incubated for 24h with peptide pulsed T2-B7 cells at an effector to target ratio of 2:1. Supematants were collected and IFN-γ secretion was measured by ELISA (standard deviation of the mean of triplicates in one of two representative experiments is shown).

(F) For characterization of the functional avidity of the TCR2.5D6, transduced lymphocytes were co-incubated with T2-B7 cells pulsed with graded amounts of MPO ₅ at effector to target ratios of 2:1. Supematants were collected 24h later and IFN-γ secretion was measured by ELISA (standard deviation of the mean of triplicates in one of two experiments is shown).

(G) Antigen specific IFN-γ release by TCR2.5D6-transduced lymphocytes was determined in response to the MPO-negative cell lines C1R and BJAB, transduced with HLA-B7 alone or HLA-B7 and MPO, respectively. Cell lines transduced with both constructs were additionally pulsed with MPO ₅ as positive control, untransduced lymphocytes were used as negative control.

Figure 3 shows experimental data characterizing the reactivity of TCR2.5D6-transduced lymphocytes in response to leukemic cell lines and primary leukemia samples.

Lymphocytes either untransduced or transduced with the TCR2.5D6 (transduction rate: 19%, Figure 2D), were stimulated with target cells for 24h at an effector to target ratio of 2:1. Supematants were collected and IFN-γ secretion was analyzed by ELISA. Standard deviation of the mean of triplicates in one out of at least two representative experiments is shown.

(A) Myeloid leukemia cell lines with different MPO expression (Figure 9 A and B) were transduced with the retroviral construct HL4-B7-P2A-eGFP. Cell lines were additionally pulsed with MPO ₅ at a concentration of 1 μΜ as positive controls.

(B) Primary samples of patients with MPN and AML were pretreated as described in Example 2. MPN1, MPN2, MPN5, MPN6 and AML6 express MPO and HLA-B7, MPN3 expresses MPO but not HLA-B7 and AML2 does express HLA-B*07:04 but not MPO (Figure 9 C and D). As positive control, cells were pulsed with MPO ₅ at a concentration of ΙμΜ. Reactivity of TCR2.5D6-transduced lymphocytes in response to colony forming leukemic cells was analyzed by CFU assays. Therefore, CD34 ⁺ cells (500 cells within a PBMC bulk population) of the leukemic sample MPN2 (MPO ⁺ , HLA-B7 ⁺ ) (C) and MPN3 (MPO ⁺ , HLA-B7 ^" ) (D) were incubated with untransduced (black circles) or TCR2.5D6-transduced (open squares) at different effector to target ratios for 15 minutes and plated in duplicates in methylcellulose medium. Colonies were counted 14 days later under a light microscope. The mean of duplicates in one out of two representative experiments is shown.

Figure 4 shows experimental data indicating that TCR2.5D6-transduced CD8 ⁺ TCM (TCM: central memory T cells) increase survival in a xenogeneic murine AML model.

BRG mice were irradiated with 3.5 Gy and inoculated intravenously with lxl 0 ⁶ NB4-B7 leukemia cells 24 hours later. T cells were transferred 24h thereafter by intravenous injection of lOxlO ⁶ untransduced (n = 6) or TCR2.5D6-transduced (n = 6) CD8 ⁺ T _CM (Phenotype and functionality: Figure 10). Control mice (n = 5) received 200μ1 PBS only. Continuous support of human IL-15 was provided by biweekly intraperitoneal injection of 15xl0 ⁶ IL-15 producing γ- irradiated NSO cells.

(A) Infiltration by human CD45 ⁺ CD3 ^" CD8 ^" tumor cells in the bone marrow was analyzed using flow cytometry. Statistical analysis was done using Mann- Whitney test (*P < 0.0247, **P = 0.0043).

(B) Kaplan-Meier curves are shown for all three groups. Statistical analysis was done between animals receiving untransduced CD8 ⁺ TCM and CD8 ⁺ TCM transduced with TCR2.5D6 using the Mantel-Cox test (**P = 0.0048).

(C) Diseased mice were sacrificed and the percentage of eGFP-expressing, human CD45 ⁺ cells was analyzed in resected tumors.

Figure 5 shows experimental data examining the hematotoxicity of lymphocytes transduced with the TCR2.5D6.

(A) Lymphocytes transduced with TCR2.5D6 (transduction rate: 19%, Figure 2D), were tested for potential on-target toxicity. IFN-γ secretion was measured by ELISA after stimulation with different target cells at effector to target ratios of 2:1 for 20h. Standard deviation of the mean of triplicates in one out of at least two representative experiments is shown. Recognition of PBMC subsets of an HLA-B7 positive healthy donor was analyzed by incubation of TCR2.5D6- transduced or untransduced lymphocytes with isolated CD3 ⁺ , CD56 ⁺ , CD19 ⁺ , CD14 ⁺ cells and granulocytes (MPO-expression data: Figure 15 A and B). As positive control, cells were pulsed with Ι μΜ MPO ₅ . (B) Reactivity of TCR2.5D6-transduced lymphocytes against HLA-B7 , CD34 ⁺ hematopoietic stem cells was assessed in CFU assays. CD34 ⁺ cells (500 cells within a PBMC bulk population) were incubated at different effector to target ratios with untransduced or TCR2.5D6-transduced lymphocytes for lh. Cells were plated in duplicates in methylcellulose medium. Colonies were counted 14 days later under a light microscope. The mean of duplicates in one out of two representative experiments is shown.

(C) As positive control, target cells were pulsed with ΙμΜ MPO ₅ .

Figure 6 shows experimental data studying off-target reactivity of lymphocytes transduced with the TCR2.5D6

Lymphocytes transduced with TCR2.5D6 (transduction rate: 19%, Figure 2D), were tested for potential off-target toxicity. IFN-γ secretion was measured by ELISA after stimulation with different target cells at effector to target ratios of 2:1 for 20h. Standard deviation of the mean of triplicates in one out of at least two representative experiments is shown.

(A) Peptide dependent and independent allo-HLA reactivity was measured by IFN-γ secretion after stimulation of TCR2.5D6-transduced or untransduced lymphocytes with LCL expressing common HLA alleles (Table 4). Binding of MPO ₅ to diverse HLA alleles was investigated by pulsing LCL with 1 μΜ MPO ₅ . LCL1 is HLA-B7 ⁺ and served as a positive control.

(B) Lymphocytes untransduced or transduced with TCR2.5D6 were stimulated with C1R cells transduced with HLA-B*42:01. Target cells were additionally transduced with MPO and pulsed with MPO ₅ peptide.

(C) Recognition of HLA-B7-transduced, non-myeloid cell lines SKOV-3, 888mel, SW480, 143TK ^" , SKBR-3 (transduction rate 25-50% without sorting and cloning) and primary HRGEC (HLA-B7 ) by TCR2.5D6-transduced lymphocytes was tested (MPO protein expression: Figure 15 C).

(D) To determine the MPO ₅ amino acid residues critical for recognition by TCR2.5D6, alanine- and threonine-variants of MPO ₅ were loaded onto the HLA-B7 ⁺ cell line LCL1 at a concentration of ΙμΜ. The wildtype MPO ₅ peptide was used as a positive control, unpulsed target cells as a negative control. (E) 65 naturally presented HLA-B7 ligands were divided into 6 peptide pools. The pools were loaded onto T2-B7 cells at a final concentration of 1 μΜ for each peptide. T2-B7 cells pulsed with MPO ₅ were used as a positive control.

Figure 7 shows a schematic overview of the stimulation procedure for MPO-specific T cells in the single HLA-mismatched setting.

Schematic overview of the single HLA-mismatched T cell priming protocol for expansion of peptide specific T cells recognizing naturally HLA-presented MPO peptides. 3d DCs of a HLA- B7- blood donor were electroporated with HLA-B7-P2A-eGFP in vitro transcribed (ivt)-RNA, pulsed with MPO ₅ and used for priming of naive T cells from the same donor. Cells were restimulated with MPO ₅ pulsed C1R-B7 cells on day ten. Ten days later, expansion of MPO ₅ specific T cells was analyzed with an MPO ₅ specific multimer and multimer positive cells were sorted on a MoFlo high performance cell sorter.

Figure 8 shows a detailed characterization of the mouse model used in Figure 4.

(A) Schematic overview of the xenogenic mouse model used in Figure 4. 6 - 8 weeks old BRG mice were irradiated with 3.5Gy and inoculated i.v. with 1 x 10 ⁶ HLA-5*07.02-P2A-eGFP- transgenic NB4 cells (NB4-B7) the day after. 10 x 10 ⁶ TCR2.5D6-transduced CD8 ⁺ T _CM were injected i.v. one day later; control mice received untransduced CD8 ⁺ T _CM or PBS. In order to improve engraftment and persistence of human T cells mice were inoculated with 15 x 10 ⁶ human IL-15 producing NSO cells i.p. twice a week. Mice were sacrificed when suffering from leukemic disease.

(B) Typical manifestation of the disease in the lymphnodes of a mouse sacrifized on day 30.

(C) [i8F]-FDG PET of a control mouse that received 200μ1 PBS i.v. (i) or a mouse that received lxl0 ⁶ NB4-B7 cells (ii) on day 30.

(D) Representative hematoxylin and eosin, IHC-MPO and human CD45/CD8 FACS-stains of tumors from a mouse that did not receive T cells (i), untransduced (ii) or TCR2.5D6-transduced TcM(iii).

Figure 9 shows experimental data obtained from an expression analysis of MPO in HLA-B7- transduced leukemia cell lines and primary leukemia samples used for stimulation of TCR2.5D6- transduced lymphocytes. (A) Expression of MPO on the protein level in leukemia cell lines was analyzed by Western Blot. mRNA expression of MPO relative to PBMC was analyzed by qPCR in leukemia cell lines

(B) and primary leukemic samples (C). Ct values were normalized against the geometric mean of three housekeeping genes (GAPDH, HPRT1, HMBS) using the ddCT method. (D) Expression of MPO in primary leukemia samples of MPN and AML patients was determined by intracellular staining of MPO and analysis on a flow cytometer. MPN samples were treated for six days with lOOng/ml SCF and FLT3-ligand and overnight with 500IU/ml IFN-γ. Samples of patients with AML were treated overnight with 500IU/ml IFN-γ.

Figure 10 shows experimental data to characterize the phenotype and functionality of injected CD8 ⁺ T _C M for the in vivo experiment shown in Figure 4.

For mouse experiments, CD8 ⁺ CD62L ⁺ CD45RA ^" T _CM were isolated on day 0 by magnetic bead cell depletion and selection. Cells were activated for two days in the presence of CD3/CD28 beads and 50IU/ml IL-2 and transduced with TCR2.5D6 virus supernatant or left untreated.

(A) Cells were analyzed for expression of the surface markers CD4, CD8, CD45RA and CD62L. Expression of the transgenic TCR chains was analyzed by staining with the MPO ₅ specific HLA multimer and an antibody directed against the murinized regions of the TCR.

(B) Functionality of the TCR2.5D6-transduced (transduction rate: 47%) or the untransduced CD 8 T _CM cells was tested. Lymphocytes were incubated with T2-B7 cells pulsed with MPO ₅ or an irrelevant peptide and NB4-B7 leukemia cells as well as NB4-B7 cells were pulsed with 1 μΜ MPO ₅ , as positive control, at an effector to target ration of 2:1. Cells were incubated for 20h and IFN-γ secretion was measured in the supernatant by ELIS A. Standard deviation of the mean of triplicates is shown.

(C) Cytotoxic activity was assessed in the same preparation as Figure 10 B 52h later by determination of the percentage of eGFP-expressing target cells in the lifegate.

Figure 11 shows the gating strategy for the analysis of eGFP expression of human CD45 ⁺ CD8 ^" cells in bone marrow and resected tumors in mice from the experiment shown in Figure 4

Single cell suspensions were stained with 7-AAD for exclusion of dead cells. Cells were further stained with an antibody specific for human CD45 and CD8 and analyzed for their eGFP expression. Percentage of cells in the lifegate is shown. (A) Bone marrow isolated out of the femur of a mouse that did not receive T cells. (B) Single cell suspensions of pooled tumors of an individual mouse that received untransduced T cells.

Figure 12 portrays experimental data showing the loss of eGFP and HLA-B7 but not MPO expression in the tumor of a mouse (Figure 4) treated with TCR2.5D6-transduced CD8 ⁺ TCM- The tumor of a mouse that was treated with TCR2.5D6-transduced CD8 ⁺ TCM was resected, a single cell suspension was obtained and cells were stained for hCD45, HLA-B7 and intracellular MPO. Viable, human CD45 ⁺ , eGFP ^" cells were analyzed for HLA-B7 and MPO expression by flow cytometry.

Figure 13 shows experimental data indicating organ-infiltration of human T cells in the xeno genie murine AML model (Figure 4).

Diseased mice were sacrificed and organs were analyzed for T cell infiltration by flow cytometry. The gating strategy for infiltration of human T cells into the spleen of a mouse that received TCR2.5D6-transduced CD8 ⁺ TCM is shown in (A), for a mouse that received untransduced CD8 ⁺ T _CM in (B). Infiltration of human CD45 ⁺ CD3 ⁺ CD8 ⁺ (C) or CD45 ⁺ CD3 ⁺ CD8 ⁺ TCRm ⁺ (D) T cells in bone marrow, tumor, lung and spleen was determined in all groups. Percentages of lifegate are shown. Data of groups„CD8 ⁺ TCM untransduced" and „CD8 ⁺ TcM TCR2.5D6" were compared using Mann- Whitney test (*P = 0.0117, **P < 0.0087).

Figure 14 shows anti-leukemic reactivity of TCR2.5D6-transgenic unselected lymphocytes in vivo.

(A) Schematic overview of the experimental process. BRG mice were irradiated with 3.5 Gy and inoculated with 5xl0 ⁶ NB4-B7 cells via the tail vein. Three days later, no lymphocytes, lOxlO ⁶ untransduced or TCR2.5D6-transduced lymphocytes as depicted in Figure 2D and E were injected i.v. Systemic support of human IL-15 was applied by biweekly i.p. injection of 15xl0 ⁶ irradiated NSO-IL15 cells. Mice were sacrificed when suffering from disease with respective criteria as written in Example 2.

(B) Bone marrow of sacrificed mice was isolated and the percentage of CD45 ⁺ eGFP ⁺ cells was determined by flow cytometry. Statistical analysis was done using the Mann- Whitney test (**P< 0.0025).

(C) Kaplan-Meier curves are shown for all three groups. (D) Diseased mice were dissected between day 21 and 34, visible tumors were resected and further prepared to obtain single cell suspensions. Pooled suspensions from each individual mouse were analyzed for their percentage of hCD45 ⁺ CD8 ^" cells expressing eGFP by flow cytometry.

Figure 15 shows experimental data on the expression of MPO in primary hematopoietic cells and non-hematopoietic cell lines.

(A) mRNA expression of MPO relative to PBMC was analyzed by qPCR in PBMC subsets and granulocytes. Ct values were normalized against the geometric mean of three housekeeping genes (GAPDH, HPRT1, HMBS) using the ddCT method. Expression of MPO in the bone marrow compared to PBMC is shown as a positive control.

(B) Expression of MPO in negatively isolated PBMC subsets and granulocytes was analyzed by intracellular staining of MPO and flow cytometry analysis. All isolated PBMC subsets were stained with an anti-human MPO antibody and a subset specific surface marker. Granulocytes were identified according to a high granularity in the side scatter.

(C) Expression of MPO in cell lines of non-hematopoietic origin was analyzed by Western Blot. Lysates of HL-60-B7 cells were loaded as a positive control.

Figure 16 shows additional data for stem cell cytotoxicity and reactivity against alanine- and threonine-variants

(A) Reactivity of TCR2.5D6-transduced lymphocytes against HLA-B7+, CD34+ hematopoietic stem cells was assessed in CFU assays. CD34+ cells (500 cells within a PBMC bulk population) were incubated at different effector to target ratios with untransduced or TCR2.5D6-transduced lymphocytes for 24h. Cells were plated in duplicates in methylcellulose medium. Colonies were counted 14 days later under a light microscope. The mean of duplicates is shown.

Alanine and threonine exchange-variants of the MPO ₅ peptide were analyzed for their recognition by TCR2.5D6-transduced lymphocytes in context with JiLA-B*07:02. Therefore, the HLA-B7 positive cell lines LCL11 (B) and LCL12 (C) were pulsed with ΙμΜ of the respective peptide and co-incubated with untransduced or TCR2.5D6-transduced lymphocytes at effector :tar get ratios of 2:1. Supematants were analyzed for the amount of IFN-γ via ELISA 24h later. Standard deviation of the mean of triplicates in one out of three experiments is shown. Figure 17 Binding specificity and affinity of aTCRmu-IgG and aTCRrnu-F(ab') ₂ and their functional modulation of TCR2.5D6-transduced cells.

(A) Flow cytometry based ¾ determination of aTCRmu-IgG and aTCRmu-F(ab') ₂ using TCR2.5D6-transduced Jurkat76 cells. MFI values for indicated concentrations of aTCRmu-IgG or aTCRmu-F(ab') ₂ staining are depicted.

(B-C) Apoptotic cells (left graph) and IFNy production (right graph) after incubation of TCR2.5D6-transduced T _CM with indicated concentrations of aTCRmu-IgG and isotype-IgG (B) or with indicated concentrations of aTCRmu-F(ab') ₂ and isotype-F(ab') ₂ (C) for 12h. Non- transduced T _CM (white circles/squares) incubated with aTCRmu-IgG (B) or aTCRmu-F(ab') ₂ (C) were used as additional controls.

(D) Percentage of engrafted 2.5D6-iRFP-positive T cells in indicated organs of NSG mice 6 days post intravenous injection of aTCRmu-IgG, isotype-IgG, TCRmu-F(ab") ₂ or isotype-F(ab ^, ) ₂ . BM = bone marrow. Mean ± s.d. of %iRFP+ cells of total viable (CD45+CD3+CD5+) T cells engrafted in animals treated with aTCRmu/isotype-IgG and aTCRmu-F(aV) ₂ (n=4) and isotype- F(ab") ₂ (n=3) is shown.

Figure 18 Functionality of TCR2.5D6-transduced T _CM after labeling with

F(ab') ₂ in vitro and imaging of intratumoral injected TCR2.5D6-transduced T _CM after application of ⁸⁹ Zr-aTCRmu-F(ab') ₂ in vivo.

(A) IFNy secretion of TCR2.5D6-transduced T _CM after co-cultivation with ML2-B7 is shown. Co-incubation of T _CM and tumor cells was performed either directly (Oh) or 48h after labeling with indicated concentrations of ⁸⁹ Zr-aTCRmu-F(ab') ₂ or non-radioactive aTCRmu-F(ab') ₂ . Mean ± s.d. of triplicates is depicted.

(B) 3D-PET MIP and axial PET/CT images of mice 48h post intravenous injection

aTCRmu-F(ab') ₂ . TCR2.5D6-transduced T _CM had been previously injected into the ML2-B7 tumors and non-transduced T _CM into ML2-WT tumors in these mice (experimental design in Figure 23). Injection of PBS into tumors was used as negative control. Red arrows indicate the position of ML2-B7 tumors and white arrows the position of ML2-WT tumors. Dotted lines, (a) and (b), show the position of axial PET/CT images. Scale bar = 0-15%ID/g; L=Liver; K=Kidney.

(C) Quantitative analysis of PET-images. Mean ± s.d. of %ID/g in defined ROI is depicted. (D) Quantitative evaluation of tumor or organ to blood ratios 48h post intravenous injection of ⁸⁹ Zr-aTCRmu-F(ab') ₂ by ex vivo biodistribution. Mean ± s.d. of %ID/g ratio is depicted.

(B-D) Representative experiment using n=4 animals for T _CM and n=2 animals for PBS.

Figure 19 Investigation of dynamics of tumor rejection mediated by TCR2.5D6-transduced T _CM to determine biologically critical imaging time points in a ML2-based myeloid sarcoma model.

Tumor size of subcutaneous ML2-B7 tumors (A) and ML2-WT tumors (B) at indicated days post intravenous T _CM injection is shown. Mean ± s.d. of tumor size is indicated in mm ² of n=6 animals. Flow cytometry analyses of single cell suspensions from tumors and organs were performed at day 10 following T cell injection. The percentage of viable human T cells (C) and percent TCR2.5D6-transduced T cells of viable T cells (D) within the ML2-B7 or ML2-WT tumors, spleen and lung is shown. Mean ± s.d. of viable T cells (%CD45+ CD3+CD5+) or TCR- transduced T cells (%CD5+TCRmu+) of viable T cells is depicted for n=6 animals; Mann- Whitney Test: **p<0.01.

Figure 20 Tracking of intravenously injected TCR-transduced T _CM within ML2-B7 tumors by ⁸⁹ Zr-aTCRmu-F(ab') ₂ .

(A) Representative 3D-PET (ΜΓΡ) and axial PET/CT images 48h post intravenous injection of ⁸⁹ Zr-aTCRmu-F(ab') ₂ into tumor bearing mice that have been treated intravenously with TCR2.5D6-transduced, non-transduced T _CM or PBS (experimental design in Figure 24B). Red arrows indicate the signal at the ML2-B7 tumors of animals injected intravenously with TCR2.5D6-transduced T _CM - Dotted lines and (a) show the position of axial PET/CT images. Scale bar = 0-15%ID/g; L=Liver; K=Kidney.

(B) Quantitative analysis of PET/CT images of ML2-B7 and ML2-WT tumors. Mean ± s.d. of %ID/g of defined ROI is depicted for n=5 animals in each group treated with T _CM and n=2 treated with PBS.

(C) Quantitative evaluation of tumor or organ to blood ratios 48h post injection of ⁸⁹ Zr-aTCRmu- F(ab') ₂ . by ex vivo biodistribution Mean ± s.d. of %ID/g ratio is depicted for n=5 animals in each group treated with T _CM and n=2 treated with PBS.

(D) Representative data of autoradiography are shown. (A-D) Representative data of one out of three experiments including animals treated with TCR2.5D6 TCM (n=l l), non-transduced T _CM (n=11) and PBS (n=4). Mann- Whitney test: *p<0.05 **p<0.01.

Figure 21 Presence of aTCRmu positive T cells in ML2-B7 tumors analyzed ex vivo following PET/CT imaging.

(A, B) Ex vivo flow cytometry analyses of single cell suspensions from tumors and organs after 48h post ⁸⁹ Zr-aTCRmu-F(ab ^, ) ₂ injection. The percentage of viable T cells (%CD45+ CD3+CD5+) (A) and of TCR2.5D6-transduced T cells (%CD5+ TCRmu+) of all viable T cells

(B) is shown. Mean ± s.d. of mice injected with TCR2.5D6-transduced T cells (n=5), non- transduced T cells (n=5) and PBS (n=2) is depicted.

(C) RT-PCR of the TCRmu gene using gDNA isolated from single cell suspensions of tumors and organs. gDNA of ML2-B7 and non-transduced T _CM served as negative control, gDNA of TCR2.5D6-transduced T _CM as positive control. Percentage of TCRmu molecules normalized to the number of human TCR (huTCR) molecules is depicted.

(D) H/E staining (upper panel) of ML2-B7 and ML2-WT tumors; bar=40(μm. Corresponding immunohistochemistry with anti-human CD5 of tumor-infiltrating T cells at two different areas of the ML2-B7 and ML-2-WT tumor (middle and lower panel) is shown; bar=5(μm. Mann- Whitney Test: *p<0.05 **p<0.01.

Figure 22 Differential distribution of Zr-aTCRmu-F(ab ^, ) ₂ signals within ML2-B7 tumors of mice injected with 2.5D6TCR-transgenic T _CM .

(A) Representative 3D-PET (MIP) pictures of ML2-B7 tumors 48h post intravenous injection of

SO

Zr-aTCRmu-F(ab') ₂ are shown (experimental design in Figure 24B). Definition of tumor models in group I (n=4), group II (n=4) and group III (n=3) depending on PET signal distribution and tumor size are shown. Scale bar = 0-15%ID/g; T=Tumor; KHKidney.

(B) ML2-B7 tumor development in mice injected with TCR2.5D6-transduced T _CM or non- transduced T _CM according to the classification within group I, group II and group III. Tumor size in mm at indicated days post T _CM injection is depicted. Arrows indicate the days of T _CM injection and PET/CT imaging. (C) Quantitative analysis of PET-signal of ML2-B7 tumors classified according to group classification. Mean ± s.d. of %ID of defined ROI is depicted.

(D) Quantitative analysis of PET/CT images of ML2-B7 tumors according to group classification. Mean ± s.d. of %ID/g of defined ROI is depicted.

(A-D) Data of 3 experiments are combined and include animals treated with TCR2.5D6 T _CM (n=l l).

Figure 23 Investigation of different imaging time points to track TCR-transduced T _CM after injection of ⁸⁹ Zr-aTCRmu-F(ab') ₂ .

(A) Overview of the experimental setting: lxlO ⁷ TCR-transduced T _CM were injected directly into subcutaneoulsy inoculated ML2-B7 tumors and lxl 0 ⁷ non-transduced T _CM were injected into ML2-WT tumors. In control mice, PBS was injected into both tumors. ⁸⁹ Zr-aTCRmu-F(ab') ₂ was subsequently injected intravenously and PET/CT imaging was performed 48h (B), 24h (C) or 12h (D) post injection (p.i.).

Representative 3D-PET (MIP) images 48h (B), 24h (C) or 12h (D) p.i. of ⁸⁹ Zr-aTCRmu-F(ab') ₂ with marked tumors is shown on the left graph. Scale bar: 0-15%ID/g. Quantitative ROI analysis of ML2-B7 and ML2-WT tumors of PET images 48h (B), 24h (C) or 12h (D) p.i. of ⁸⁹ Zr- aTCRmu-F(ab') ₂ is depicted on the middle graph. Mean ± s.d. of %ID/g in defined ROI is depicted. Graphs on the right side show tumor to blood ratio of %ID/g following ex vivo biodistribution analysis 48h (B), 24h (C) or 12h (D) post injection of ⁸⁹ Zr-aTCRmu-F(ab') ₂ . Mean ± s.d. of %ID/g ratio is depicted. Analysis has been performed with (B) T _CM (n=3) and PBS (n=2), (C) T _CM (n=6) and PBS (n=2), (D) T _CM (n=5) and PBS (n=2). R=right, L=left, non- transd. = non-transduced.

Figure 24 Establishment of a clinically relevant ML2 -based myeloid sarcoma model and determination of biodistribution of ⁸⁹ Zr-aTCRmu-F(ab') ₂ within this model.

(A) Overview of the experimental setting. NSG mice were inoculated subcutaneoulsy with lxlO ⁷ ML2-B7 cells into the right and ML2-WT cells in to the left flank followed by IGy total body irradiation (TBI). Subsequently, TCR2.5D6-transduced T _CM , non-transduced T _CM or PBS were adoptively transferred. Irradiated (80Gy) human interleukin-15 producing NSO-cells were injected intraperitoneally three times per week. (B) Overview of the experimental setting as used for imaging experiments: NSG-mice were treated as described in (A). Three days after TCM injection, ⁸⁹ Zr-aTCRmu-F(ab') ₂ was injected intravenously followed by PET/CT imaging after 48h.

(C) Ex vivo biodistribution analysis of ⁸⁹ Zr-aTCRmu-F(ab') ₂ in indicated organs 48h post injection. %ID/g ± s.d. is depicted for n=5 animals in each group treated with TCM and n=2 treated with PBS. Representative data of one out of three experiments including animals treated with TCR2.5D6 T _CM (n=l l), non-transduced TCM (n=l l) and PBS (n=4). Mann- Whitney test: **p<0.01.

Figure 25 Analysis of tumor volume and aTCRmu-F(ab') ₂ uptake in ML2-B7 tumors according the classification into group I-III.

(A) Tumor volume determined in vivo by ROI analysis of PET/CT images 48h post injection of aTCRmu-F(ab') ₂ . Mean ± s.d. of cm ³ of defined ROI is shown.

(B) Tumor weight of ML2-B7 tumors determined ex vivo after PET/CT imaging. Mean ± s.d. of ML2-B7 tumor weight in grams is depicted,

(C) Determination of ex vivo uptake of ML2-B7 tumors. Mean ± s.d. of %ID is depicted.

(A-C) Data of 3 experiments are combined and include animals treated with TCR2.5D6 TCM (n=11).

In the following, reference is made to the examples, which are given to illustrate, not to limit the present invention.

EXAMPLES Example 1

Primary material and cell lines

Blood from healthy individuals and patients with diverse hematopoietic malignancies as well as human healthy organ tissue samples obtained from diagnostic preparations were collected after informed consent following requirements of the local ethical board and principles of the Helsinki Declaration. Patient characteristics are shown in Table 3. PBMC (Peripheral Blood Mononuclear Cells) were isolated by density gradient centrifugation using Ficoll/Hypaque (Biochrom). Granulocytes were purified from whole blood by differential centrifugation as described previously (Eggleton et al, 1989). All target cells were maintained in RPMI 1640 (Invitrogen) supplemented with Penicillin/Streptomycin (PAA) and 10% fetal calf serum (Invitrogen) unless otherwise stated. Details regarding isolation of naive T cells and TCM as well as the cell lines used in the present study can be found in Example 2. Transduction of cell lines with HLA-B7- P2A-eGFP is indicated by the additional specification "-B7" to the name of the cell line, transduction with MPO-P2A-dsRed by the additional specification "/MPO". For functional in vitro assays, CML and AML samples as well as non-lymphoid cell lines were treated as described in Example 2. Primary human renal glomerular epithelial cells (HRGEC) were purchased from ScienceCell and cultured according to the manufacturer's instructions. The hybridoma cell line HB-95 (ATCC (American Type Culture Collection)) was used for in-house production of the w6/32 antibody following standard protocols.

Immunopeptidomic identification of MPO HLA ligands

HLA I-restricted peptides were identified and analyzed by the immunopeptidomic approach as previously described (Bassani-Sternberg et al., 2010; Falk et al, 1991). After HLA I immunoprecipitation and peptide elution, peptides were analyzed by nano-HPLC (Ultimate 3000, Dionex) coupled to a linear quadruple ion trap-Orbitrap (LTQ Orbitrap XL) mass spectrometer (Thermo Fisher) equipped with a nano-electrospray ionization (ESI) source as previously described (Hauck et al., 2010), allowing for the fragmentation of peptides with charges +1 to +3.

MS/MS data were analyzed using the software tools MASCOT, Peaks and Pep-Miner and potential HLA ligands were further identified and selected as described in Example 2. To screen for HLA ligands derived from genes with suitable expression patterns for T cell targeting, gene expression was determined by database and literature research (http://genome.ucsc.edu/cgi- bin/hgBlat, http://www.biogps.org, http://www.ncbi.nlm.nih.gov/ pubmed/). For sequence validation of selected candidate ligands, synthetic counterparts (IBA) were analyzed by mass spectrometry. After normalization, spectra were matched using mMass (Niedermeyer et al, 2012).

MPO expression analysis by semi-quantitative real-time polymerase chain reaction, immunohistochemistry and Western Blot

To verify the expression profile of MPO in primary cells as well as diverse human tissue, semi- quantitative real-time PCR (qPCR), immunohistochemistry and Western Blot was conducted as described in Example 2.

Expansion of T cells specific for a HLA-B7-restricted MPO epitope

To expand T cells with specificity against the selected HLA ligands, we used a single HLA mismatch priming approach as previously published (Wilde et al., 2009) with some modifications (Figure 7 and Example 2). Expanded T cells were analyzed and sorted on day 20 using HLA multimers on a high-performance cell sorter (MoFlo; Dako) and cloned by limiting dilution.

HLA multimers and antibodies

HLA multimers were synthesized as previously reported (Knabel et al., 2002). Antibodies used for flow cytometry, western blot and immunohistochemistry are listed in the Table 5.

Retroviral TCR transfer into PBMC

The sequence of the reactive TCR2.5D6 was determined by PCR and sequencing as previously described (Schuster et al, 2007; Weigand et al., 2012). A bi-cistronic construct separated by a P2A element consisting of beta and alpha chains containing murinized constant chains and an additional disulphide bridge was generated in silico and codon-optimized (Invitrogen) as previously described (Cohen et al., 2006; Kuball et al., 2007; Scholten et al, 2006). The construct was cloned into the retroviral vector pMP71 (Fehse et al. 2002), retroviral supernatants were generated and PBMC or CD8 ⁺ T _CM were transduced as previously described (Weigand et al, 2012; Wang et al., 2012).

Functional characterization of T cell clones as well as TCR-trans genie T cells T cells were tested for their cytotoxic reactivity against ⁵¹ Cr labeled target cells in a standard 4- hour ⁵¹ Cr-release assay (Schuster et al, 2007) or by a flow cytometry-based cytotoxic assay. For cytokine detection, effector and target cells were incubated at a ratio of 2:1 for 24h. Supernatants were collected and cytokine levels were measured using IFN-γ enzyme linked immunosorbent assay (ELISA) (BD) according to the manufacturer's instructions. Alanine-variants of the MPO ₅ peptide (Genscript) were used to define the residues critical for recognition by TCR2.5D6 as described in Liang et al., 2010. The ScanProsite tool was used for identification of proteins that contain the motif "XPRWDXXRL" [SEQ ID NO. 14] as described in Linette et al., 2013. HLA- B7 ligands analyzed for off-target reactivity were synthesized on an automated peptide synthesizer EPS221 (Abimed, Langenfeld, Germany) following the 9-fluorenylmethyl- oxycarbonyl/tertbutyl (Fmoc/tBu) strategy as described (Schirle et al., 2000).

In vivo anti-leukemic reactivity of TCR2.5D6-transduced T cells mice on a BALB/c background (BRG mice) were purchased from Taconic and maintained in an animal facility. Mice were sublethally irradiated with 3.5 Gy on day 0 and lxl 0 ⁶ NB4-B7 cells were injected intravenously the day after. One day later, mice received either 200μ1 PBS, lxl 0 ⁷ untransduced or lxlO ⁷ 2.5D6TCR-transduced CD8+T _CM intravenously. To provide a systemic supply of human IL-15, 1.5xl0 ⁷ , irradiated NSO-IL-15 cells (kindly provided by Stanley Riddell) were injected intraperitoneally twice per week as described in Wang et al., 2011. Diseased mice (Criteria: Example 2) were sacrificed and analyzed for tumor load and T cell engraftment by flow cytometry after organ collection and preparation of single cell suspensions.

Hematopoietic colony forming assays

All hematopoietic progenitor assays were performed according to the manufacturer's instructions (Stem Cell Technologies; Vancouver, Canada). PBMC samples containing a total of 500 CD34 ⁺ cells were co-incubated with either TCR2.5D6-transduced or untransduced lymphocytes. Incubation was performed in 250μ1 alpha MEM medium (PAA) supplemented with 2% FCS at different effector to target ratios for 15 minutes or lh at 37°C. Subsequently, cell suspensions were mixed with 2.5ml MethoCult H4435 (Stemcell Technologies) and plated in duplicates of 1.1ml in 35mm petri dishes. After 12-14 days of incubation at 37°C/5% C0 ₂ in a humidified atmosphere, plates were scored for colony growth under a light microscope.

Example 2

Animal experiments

Health status of the mice was monitored daily by an independent, experienced research assistant in the animal facility. Mice that suffered from health problems indicated by weight loss (>10%), tumor manifestation (>1 cm), rough hair-coat, changes in breathing rate or reduced mobility were sacrificed.

MS/MS data analysis and identification of potential HLA ligands

MS/MS data was analyzed using three different data analysis tools, namely MASCOT (Matrix Science), Peaks (Bioinformatics Solutions Inc.) (Zhang et al., 2012) and Pep-Miner (IBM Haifa Research Lab) (Beer et al., 2004). Database searches were performed using the human part of the NCBI and UniProt database and the Human Short Peptide Variation Database (http://srs.bioinformatics.nl/hspv/search.php). Error tolerance was set to lOppm for parent ions and 0.5Da for fragment ions. Peptides with a length of 8-11 amino acids, a MASCOT score > 30, a Peaks -logPIO > 15 or a Pep-Miner score > 70 were analyzed for described anchor residues (http://www.syfpeithi.de/) for the respective HLA type of the patient samples.

Expression analysis of MPO

For expression analysis of MPO on the mRNA level by qPCR, total RNA was isolated or used from the Human Total RNA Master Panel II (Clontech). cDNA was synthesized by the Affinity Script Multiple Temperature Reverse Transcriptase (Agilent Technologies). qPCR was conducted on a LightCycler 480 System (Roche) using the KAPA SYBR Fast LightCycler 480 reagent (Peqlab). GAPDH, HMBS and HPRTl were applied as control genes for relative quantification which was calculated by the delta-delta Ct method (Livak et al., 2001) using the geometric mean of GAPDH, HMBS and HPRTl for normalization. Pooled RNA from PBMC of three different healthy donors was used as a reference in all qPCR experiments. MPO immunohistochemistry was done using a rabbit anti-human MPO antibody (Dako) on an automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA). Two independent observers analyzed MPO-stained slides for cytoplasmatic positivity. For expression analysis of MPO on the protein level, SDS-PAGE followed by Western Blot was performed using MPO specific (EPR4792, Epitomics) and GAPDH specific (6C5, Affinity BioReagents) antibodies for loading control.

Constructs used for testing of specificity and HLA restriction of isolated T cells and clones

To transduce the restriction element HLA-B7 and the antigen MPO (NM_000250.1) into cell lines not expressing these genes, genes were cloned into the retroviral vector MP71 together with eGFP or dsRed, respectively as bi-cistronic constructs separated by a Porcine teschovirus-1- derived peptide element (P2A). For generation of in vitro transcribed (z ^' vtj-RNA, the respective HLA molecule was cloned into the plasmid pcDNA3.1(-) (Invitrogen) together with eGFP, separated by a P2A element. eGFP and dsRed were used to follow gene-transfer efficacy and to allow untouched sorting of modified cells without the need for specific antibodies. Unless otherwise stated, transduced cells were sorted according to fluorescent dye expression and cloned by limiting dilution.

Primary cells and cell lines

The following cell lines were used for analysis of MPO specific T cell receptors: T2 (ATCC; American Type Culture Collection, Manassas, VA), C1R (Falk et al., 1995), NB4 (Cell lines service, CLS, Germany), MV4-11 (CLS), HL-60 (CLS), ML-2 (The CABRI consortium), KGla (CLS), K562 (ATCC), BJAB (kindly provided by J. Mautner), Molt4 (CLS), SKOV-3 (ATCC), 888-mel (NIH), 143TK ^" (kindly provided by Ralph Mocikat), SW-480 (ATCC), SKBR-3 (CLS) and different lymphoblastoid cell lines (LCL) (kindly provided by Steve Marsh; see also Table 4). 293 HEK embryonal kidney cells (ATCC) were used for production of retroviral supernatants. For functional in vitro assays, CML and AML samples as well as non-lymphoid cell lines were treated for 24h with 500 U/ml IFN-γ (Peprotech). Cryopreserved primary cells of CML patients were additionally treated for six days with stem cell factor (SCF) and FLT3 ligand (Peprotech) at a concentration of lOOng/ml (Volpe et al., 2007). Isolation of naive T cells and CD8 T _CM

For isolation of untouched, naive CD8 T cells, CD8 T cells were negatively isolated with the Dynabeads Untouched Human CD8 T cell isolation kit (Invitrogen) according to the manufacturer's instructions. Antigen experienced T cells were further depleted with CD45RO- and CD57-microbeads (Miltenyi). For isolation of CD8 ⁺ CD62L ⁺ CD45RA ^" T _CM , CD4 ⁺ and CD45RA ⁺ cells were first depleted followed by positive selection of CD62L cells using fluorescent antibodies (BD) and microbeads (Miltenyi).

Stimulation of naive T cells in the single HLA-mismatched setting

Monocytes of a HLA-B7 negative donor were differentiated into DC by incubation with IL-4 (20ng/ml) and GM-CSF (lOOng/ml) (Peprotech) for 48h. Cells were further matured using TNFcc (lOng/ml), IL-Ιβ (lOng/ml), IFN-γ (5000IU/ml), PGE ₂ (250ng/ml) (Peprotech) and CL075 (1 μg/ml) (Invivogen) for 24h. DC transfected with ivt-RNA encoding HLA-B7 and eGFP were pulsed with the respective peptide at a concentration of 0.1 μΜ for 2h in AIM-V medium (Invitrogen). Naive T cells from the DC donor were isolated as described above. Priming was started at an effector to target ratio of 10:1 in the presence of IL-21 (30ng/ml) (Peprotech). IL-7 and IL-15 (5ng/ml) (Peprotech) were added every two to three days. T cells were restimulated using C1R-B7 cells, pulsed with Ο.ΙμΜ of the respective peptide on day 10.

Statistics

Statistical analysis was performed using GraphPad Prism version 5.01 (GraphPad Software, Inc.). Survival data was analyzed using the Mantel-Cox test and statistical difference in infiltration of human T cells in mouse organs was analyzed using the Mann- Whitney unpaired t test. Data was considered to be significantly different when P value < 0.05, < 0.01 or < 0.001 represented in the figures as *, ** or ***, respectively. Example 3

Identification of leukemia-derived HLA lifiands and selection of MPQ as potential T cell target

All methods mentioned in this example were carried out as described in Examples 1 and 2 above.

Novel target antigens expressed and presented by myeloid leukemias that could be targeted by T cells were identified by analysis of the immunopeptidome of malignant cells obtained from patients with MPN. By mass spectrometry, 4386 unique peptides with adequate peptide length and proper anchor residues to serve as potential ligands of diverse HLA class-I molecules were identified. These peptides were then analyzed for their suitability as potential target structures for T cell mediated anti-tumor immunotherapies. Nineteen of those peptides are derived from 7 genes with presumably restricted expression in hematopoietic cells (Table 1). By three different data analysis tools, five naturally presented MPO-derived HLA ligands were detected which were restricted to 4 different HLA alleles, namely HLA-A*01:01, ULA-B*07:02, ULA-BV5.01 and HLA-B*44:02 (Table 2).

For sequence validation of MPO-derived HLA ligands, defined peptides were synthesized and reanalyzed by mass spectrometry. Resulting spectra were compared with the spectra of the naturally eluted counterpart. All peptides showed overlapping fragmentation patterns (Figure 1A). For confirmation of restricted expression of MPO to myeloid cells, qPCR and immunohistochemistry were performed. High MPO expression was observed in most MPN and AML leukemia samples as well as fetal liver and healthy bone marrow. Very low MPO gene expression was observed in mature PBMC subpopulations including CD14 ⁺ monocytes and granulocytes as well as solid organ tissues (Figure IB). Immunohistochemistry was performed on healthy organ tissues demonstrating MPO protein expression only in single myeloid cells within diverse organs with the exception of bone marrow, where expression could be detected in myeloid progenitor cells (Figure 1C). Example 4

Isolation and retroviral transfer of an allorestricted MPCVspecific TCR and confirmation of MPO-specific target cell recognition

All methods mentioned in this example were carried out as described in Examples 1 and 2 above.

Focusing on the two MPO-derived peptides presented by HLA-B7 (MPO ₂ and MPO ₅ ), it was determined whether high affinity T cells specific for these two MPO peptides presented by HLA- B7 could be isolated in the single HLA-mismatched setting.

For stimulation in a single HLA-mismatched setting, dendritic cells (DCs) of an BLA-B*07:02 negative donor were electroporated with in vitro transcribed RNA (ivt RNA) coding for eGFP. eGFP served as evidence for successful electroporation and is separated from the gene for HLA- B*07:02 by a P2A element. As can be seen from Figure 2 A, generation of single HLA- mismatched DCs was successful. Expression of the surface markers CD83 and CD86 provides evidence for successful maturation of the DCs, expression of eGFP shows that the electroporation with HLA-5*07:02P2AeGFP ivt-RNA was successful. The dendritic cells were loaded with the peptide MPO ₅ and used for stimulation of naive T cells of the same donor. The experimental procedure for priming of naive T cells is shown in Figure 7. Staining of proliferated cells after restimulation on day 20 with an MPO ₅ HLA multimer demonstrated a distinct population of peptide-specific T cells (Figure 2B). The MPO ₅ HLA multimer ^"1" T cells were sorted and cloned by limiting dilution. The resulting T cell clones were tested for their cytotoxic activity against T2-B7 cells, pulsed with MPO ₅ or an irrelevant peptide. Five clones were selected that only lysed T2-B7 cells pulsed with MPO ₅ (Figure 2C). TCR analysis revealed that all 5 T cell clones expressed the same Va and νβ chains, namely Vcd.l and νβ13.2. Murinized and optimized TCR Va and Λφ sequences (termed TCR2.5D6) were cloned into the retroviral vector pMP71 and transduced into lymphocytes of a healthy donor. Multimer binding as well as functionality after TCR-gene transfer was confirmed (Figure 2D and E). Functional avidity of the TCR was tested using T2-B7 cells pulsed with serial dilutions of MPO ₅ . As shown in Figure 2F, half maximal secretion of IFN-γ was observed at concentrations of 150pM. Specific recognition of naturally processed and presented MPO ₅ was analyzed using diverse B cell lines originally negative for MPO. In fact, C1R-B7 and BJAB-B7 cells were recognized when additionally transduced with the MPO gene verifying recognition of endogenously processed peptide (Figure 2G).

Example 5

Reactivity of TCR2.5D6-transduced lymphocytes in response to MPO-expressing leukemia cell lines as well as primary leukemia samples

All methods mentioned in this example were carried out as described in Examples 1 and 2 above.

Reactivity of TCR2.5D6-transduced lymphocytes against different leukemia cell lines and primary leukemia samples was assessed by IFN-γ ELISA. As shown in Figure 3 A, only leukemia cell lines that show expression of MPO (Figure 9 A, B), namely HL-60-B7, NB4-B7, ML-2-B7, were recognized by TCR2.5D6-transduced lymphocytes. MPO negative cell lines were only recognized when pulsed with MPO ₅ , demonstrating principal vulnerability of these cell lines by the defined TCR. No recognition by untransduced lymphocytes was observed.

Furthermore, IFN-γ production by TCR2.5D6-transduced lymphocytes in response to primary samples from patients with MPN and AML was analyzed (Figure 3B). In contrast to healthy mature myeloid cells, leukemia cells demonstrate partially high MPO-expression on both, mRNA and protein level (Figure 1 and Figure 9 C, D). Reactivity of TCR2.5D6-transduced lymphocytes could only be observed against target cells expressing both HLA-B7 and MPO (MPN1, MPN2, MPN5, MPN6 and AML6). The MPO positive but HLA-B7 negative sample MPN3 and the MPO negative, RLA-B*07:04 ⁺ sample AML2 were not recognized. Pulsing of AML2 with MPO ₅ resulted in recognition of the sample by TCR2.5D6-transduced lymphocytes indicating a potential binding of MPO ₅ also to the HLA-B7 subtype B*07:04.

To analyze the reactivity of TCR2.5D6 against colony forming leukemic precursor cells, either untransduced or TCR2.5D6-transduced lymphocytes were incubated with bulk PBMC of the HLA-B7 positive patient MPN2 at different effector to target ratios. A decrease in colony forming units (CFU) was observed, when cells of MPN2 (HLA-B7 , MPO ⁺ ) were incubated with TCR2.5D6-transduced lymphocytes at effector to target ratios of 100:1 and 1000:1 (Figure 3C), whereas no reduction was detectable when cells were incubated with untransduced lymphocytes. As a control, untransduced and TCR2.5D6-transduced lymphocytes were incubated with cells of patient MPN3 (HLA-B7 ^" , MPO ). No difference in the number of CFU was observed (Figure 3D). Taken together, these data demonstrate strong MPOs-specific reactivity of TCR2.5D6- transduced lymphocytes against leukemic cells including leukemic precursors in vitro.

Example 6

TCR2.5D6-transduced CD8 ⁺ TnM prolong survival in a xenogeneic mouse model of human AML

All methods mentioned in this example were carried out as described in Examples 1 and 2 above.

In vivo tumor reactivity of TCR2.5D6-transduced CD8 ⁺ TCM was analyzed in a xenogeneic mouse model of human AML. lxl 0 ⁶ NB4-B7 cells were injected into BRG mice, followed by injection of lOxlO ⁶ untransduced or TCR2.5D6-transduced CD8 ⁺ T _CM or PBS alone. TCR expression and functionality of injected T cells is shown in Figure 10, details regarding the mouse model are shown in Figure 8. Mice were sacrificed when suffering from health problems as described in Example 2. Infiltration of bone marrow with CD45 ⁺ CD3 ^" CD8 ^" tumor cells was significantly reduced in mice that were treated with TCR2.5D6-transgenic TCM compared to the untreated group (P = 0.0043) as well as the group that received untransduced TCM (P ⁼ 0.0247) (Figure 4A). Furthermore, a significant prolonged overall survival of mice treated with TCR2.5D6- transduced CD8 ⁺ TCM was observed compared to mice that received untransduced CD8 ⁺ TCM (P ⁼ 0.0048) with median survival of 44 days versus 34 days, respectively. In contrast, there was no significant difference in median survival between the control group that received no T cells at all compared to the group that was treated with untransduced CD8 ⁺ TCM (37 days versus 34 days, P = 0.2229) (Figure 4B). All tumors were analyzed for expression of human CD45 and eGFP. CD45 ⁺ , eGFP ^" tumors were observed in 4 out of 5 mice in the group that was treated with TCR2.5D6-transduced CD8 ⁺ TCM (Figure 4C). Concomitant loss of HLA-B7 was confirmed whereas MPO expression was preserved (Figure 12). In contrast, tumors of all animals treated with untransduced T cells or no T cells showed preserved eGFP expression (Figure 4B). As shown in Figure 13, human TCRm ⁺ T cells infiltrating bone marrow, tumor, lung and spleen could specifically be detected in the group that received TCR-transgenic T _CM - An additional experiment has been performed with clearly less favorable conditions as higher tumor load and later transfer of unselected lymphocytes with reduced TCR transduction rate (Figure 14A). Treatment still resulted in statistically significant reduction of bone marrow infiltration by leukemic cells (Figure 14B) as well a trend to improved survival (Figure 14C). In these animals, loss of GFP expression has not been observed (Figure 14D). Taken together, these data demonstrate in vivo efficacy of TCR2.5D6-transduced T _CM against leukemic cells and argue for a potent effect of this TCR against tumors with expression of both MPO and HLA-B7.

Example 7

On-target reactivity of lymphocytes transduced with the MPCs reactive TCR2.5D6

All methods mentioned in this example were carried out as described in Examples 1 and 2 above.

As it has been previously shown that on-target reactivity may induce unpredicted serious side effects, potential reactivity of TCR2.5D6-transduced lymphocytes against a number of different target cells including different HLA-B7 ⁺ hematopoietic cells was investigated. As shown in Figure 1 and Figure 15 A and B, granulocytes as well as monocytes demonstrate relevant MPO protein but very low mRNA expression. Regarding recognition by TCR2.5D6-transduced lymphocytes, IFN-γ production was only detected at very low levels in co-cultures of healthy hematopoietic cells with TCR2.5D6-transduced lymphocytes unless the target cells were pulsed with MPO ₅ (Figure 5 A). In addition, the reactivity of TCR2.5D6-transduced lymphocytes against healthy, CD34 ⁺ hematopoietic stem cells of a HLA-B7 ⁺ donor was investigated in CFU assays. As shown in Figure 5B, the potential for formation of CFU by granulocyte-colony-stimulating factor (G-CSF) mobilized HSC was not reduced when HSC were incubated for 1 hour before plating in methylcellulose medium with TCR2.5D6-transduced lymphocytes compared to untransduced lymphocytes. Similar results were obtained when HSC were incubated for 24 hours with TCR2.5D6-transduced or untransduced lymphocytes (Figure 16A). In contrast, pulsing of target cells with MPO ₅ resulted in a strong reduction in CFU when incubated with TCR2.5D6- transduced but not untransduced lymphocytes (Figure 5C). These data indicate the absence of reactivity of TCR2.5D6 against mature myeloid cells as well as normal HSC.

Example 8

Off-target reactivity of lymphocytes transduced with the MPOj-specific TCR2.5D6

All methods mentioned in this example were carried out as described in Examples 1 and 2 above.

There have been reports in the literature of fatal side effects due to unpredicted off-target reactivity after transfer of tumor-reactive transgenic T cells containing an affinity-enhanced TCR. Although TCR2.5D6 was not affinity-enhanced, we sought to screen for peptide-dependent or independent recognition of diverse common HLA molecules. Therefore, different lymphoblastoid cell lines (LCL) were tested (Table 4) either unpulsed or pulsed with MPO ₅ (Figure 6A). LCL1 is HLA-B7 ⁺ and served as positive control. No reactivity against cell lines was observed without pulsing of MPO ₅ . Following peptide pulsing, reactivity against LCL6 was observed (Figure 6A). In silico analysis of binding motifs for HLA molecules expressed by LCL6 revealed a potential binding of MPO ₅ to ULA-B*42:01 (HLA-B42). Therefore, the cell lines C1R-B42 as well as C1R-B42/MPO were generated. CIR cells were only detected after transduction of both, HLA-B42 and MPO, verifying on-target reactivity against MPO ₅ also when presented by HLA-B42 (Figure 6B). Furthermore, off-target reactivity against different cell lines of non-hematopoietic origin as well as primary HRGEC not expressing MPO was tested (expression data: Figure 15C). As shown in Figure 6C, reactivity of TCR-transgenic lymphocytes could only be observed after pulsing of target cells with MPO ₅ , further strengthen the high specificity of the TCR2.5D6. In order to investigate potential peptide promiscuity of the TCR, experiments with alanine- and threonine-variants of MPO ₅ loaded onto the HLA-B7 ⁺ cell line LCL1 were performed. As shown in Figure 6D, the residues at position 2, 3, 4, 5, 8 and 9 seem to be essential for recognition by TCR2.5D6. Similar results have been observed using two other HLA-B7 ⁺ LCL. In silico analysis using the ScanProsite tool revealed MPO being the only human protein containing the motif "XPRWDXXRL". The same has been observed when the HLA-B7 ⁺ cell lines LCL11 and LCL 12 were used as target cells (figure 16B and C). Finally, crossreactivity was tested against 65 peptides, divided into 6 pools, which have been eluted from HLA-B7 molecules. As shown in Figure 6E, no reactivity could be observed at all, except against MPO ₅ . Thus, off-target reactivity could not be observed by these experiments.

Example 9

9.1 Materials and Methods

Primary material and cell lines

Blood from healthy donors was collected for isolation of peripheral blood mononuclear cells after informed consent following requirements of the local ethical board and principles of the Helsinki Declaration. The following cell lines were used: ML2 (The CABRI consortium), NSO- IL15 cells (kindly provided by S.R. Riddell) (27), Jurkat76-CD8a (28) and the hybridoma H57- 597 (HB-218, ATCC).

Conjugation and zirconium- 89 ( ⁸⁹ Zr)-labeling of aTCRmu-F(ab') ₂

The p-isothiocanatobenzyl-derivative of desferoxamine (DFO-Bz-NCS, Macrocyclics, Inc.) was used as a bifunctional chelator for ⁸⁹ Zr and was added in a 3 -fold molar excess to the aTCRmu-F(ab') ₂ followed by purification by size exclusion chromatography (Sephadex G-25 M,

PD10 column, GE Healthcare). The labeling of aTCRmu-F(ab') ₂ with ^oy Zr was performed based on the protocol of Perk et al. (2010) with slight modifications.

Positron emission tomography/computer tomography (PET/CT) Imaging

Mice were anesthetized and imaged with the Inveon small animal PET/CT scanner (Siemens) after indicated time points following injection of Zr-aTCRmu-F(ab')2 (0,7-l,5MBq corresponding to 10-20μg). Static PET emission images were acquired for 30min and reconstructed using OSEM-3D algorithm. CT images were reconstructed using a modified Feldcamp algorithm and PET and CT images were fused and analyzed using the Inveon Research Workplace (Siemens). The data was corrected for attenuation and radioactive decay. Tracer uptake was calculated as percentage of injected dose per gram of tissue (%ID/g, lcc=lg). For quantification, a region of interest (ROI) encompassing the tumor was drawn based on CT images, transferred to the PET data and mean values (%ID/g) were determined using a threshold of 50% of maximal uptake inside the tumor region. Activity in the whole tumor was calculated by multiplying the measured mean by region volume.

Bio distribution analysis

Mice were sacrificed 48h after ⁸⁹ Zr-aTCRmu-F(ab') ₂ administration. Blood and organs were collected, weighted and radioactivity was counted against a standard of known activity in a gamma-counter (2480Wizard2, PerkinElmer). Tumor and organ uptake was calculated as %ID/g or percent injected dose (%ID) and expressed as ratio to blood uptake.

Autor adio graphy

Sections (20μηι) of tumors and organs were exposed to a phosphorimaging plate (Fujifilm) for 48h followed by detection by a Phosphor-imager (CR35BIO, Durr-Biomedical) and analyzed using AID A Image analyzer software.

Statistics

Data are presented as mean ± standard deviations (s.d.). Statistical analysis of results was performed using GraphPad Prism software version 5.01 using Mann- Whitney test as indicated in Figure legends.

9.2 Results

aTCRmu-IgG and aTCRmu-F(ab')2 bind specifically and with high affinity to TCR- transduced cells but show different impact on functionality of central memory T cells (TCM)

Murinization of TCR domains has been proposed for general transgene optimization in order to reduce the risk for mispairing of introduced and endogenous TCR (Cohen et al., 2006). The hamster aTCRmu antibody (H57-597) specifically recognizes murine TCR and engineered TCR constructs harboring a murine constant beta domain (Kubo et al., 1989; Kuball et al., 2009). For establishment of an imaging model of TCR-transgenic T cells harboring such modification, we used the human-derived leukemia-reactive TCR2.5D6 recognizing specifically a myeloperoxidase-derived peptide (MPO ₅ ) in the context of HLA-B7. Human CD8-enriched T _CM were retrovirally transduced with TCR2.5D6 and transgenic TCR expression was confirmed by aTCRmu and multimer staining (data not shown). For in vivo imrnuno-imaging of defined T-cell populations, high affinity of the antibody and conservation of T cell functionality are essential. Flow cytometry-based binding analyses using TCR2.5D6-transduced Jurkat76 cells revealed a comparable dissociation constant (¾) in the nanomolar range for the full antibody (aTCRmu- IgG) as well as its F(ab") ₂ derivate (Figure 17A). Direct antibody binding to the TCR, however, has been demonstrated to impact on functionality of murine T cells (Kubo et al., 1989; Drobyski et al., 1996; Nishimura et al., 1994; Kummer et al., 2001). We therefore analyzed the influence of aTCRmu-IgG or aTCRmu-F(ab') ₂ on transduced T _CM - Incubation of TCR-transduced T _CM with aTCRmu-IgG for 12h showed a dose-dependent increase of apoptotic cells compared to non-transduced T _CM or isotype-IgG control treatment (Figure 17B). Similarly, we detected a dose-dependent secretion of Interferon-γ (IFNy) by TCR2.5D6-transduced T _CM in contrast to non-transduced T _CM or isotype-IgG (Figure 17B). Co-incubation of TCR2.5D6-transduced T _CM with aTCRmu-F(ab') ₂ did neither show an enhancement of apoptosis nor a higher IFNy- production of treated cells (Figure 17C). To analyze the functional impact of aTCRmu-IgG and aTCRmu-F(ab') ₂ in vivo, T _CM were transduced with a vector containing TCR2.5D6 linked to the near-infrared red fluorescent protein (iRFP) and intravenously injected into NOD.Cg- Prkdc ^scid I12rg ^talwj 7SzJ (NSG)-mice. We observed reduced percentages of engrafted TCR- transgenic T cells in different organs after intravenous application of aTCRmu-IgG compared to the isotype control which was not observed for aTCRmu-F(ab ^A ) ₂ (Figure 17D).

Incubation of TCR-transgenic T _CM with ⁸⁹ Zr-labeled aTCRmu-F(ab') ₂ has no impact on T cell function in response to specific target cells

Due to the high binding affinity but lack of functional modulation of transgenic T cells by aTCRmu-F(ab') ₂ , we conjugated the aTCRmu-F(ab') ₂ with DFO-Bz-NCS followed by labeling with ⁸⁹ Zr ( ⁸⁹ Zr-aTCRmu-F(ab') ₂ ). To determine a potential functional impairment by exposure to ⁸⁹ Zr, T _CM cells were labeled in vitro by different concentrations of ⁸⁹ Zr-aTCRmu-F(ab') ₂ . Unbound ⁸⁹ Zr-aTCRmu-F(ab') ₂ was removed and labeling efficiency was determined (data not shown). Subsequently, IFNy secretion of labeled T _CM in response to ML2-wildtype (ML2-WT) cells transduced with HLA-B*0702 (ML2-B7) was determined. Directly after labeling, IFNy secretion of ⁸⁹ Zr-aTCRmu-F(ab') ₂ -labeled TCR-transduced T _CM is comparable to TCR- transduced T _CM incubated with non-radioactive aTCRmu-F(ab') ₂ and similar results were obtained at 48h post labeling and exposure to Zr (Figure 18 A).

In vivo detection of TCR-transduced T _CM injected within the tumor using ⁸⁹ Zr-aTCRmu- F(ab') ₂

To investigate the feasibility to visualize TCR-transduced T _CM by Zr-aTCRmu-F(ab') ₂ , we developed an in vivo model of myeloid sarcoma by subcutaneous inoculation of ML2-WT cells on one flank and ML2-B7 cells on the other flank of C.Cg-Rag2 ^tmlFwa I12rg ^tmlSug /JicTac (BRG)- mice. Transgenic T _CM were directly injected in the ML2-B7 tumor and non-transduced T _CM were inoculated in the ML2-WT tumor followed by intravenous injection of ⁸⁹ Zr-aTCRmu-F(ab") ₂ (Figure 23A). PET/CT imaging was performed at different time points post injection. 48h post injection of ⁸⁹ Zr-aTCRmu-F(ab") ₂ , we observed a strongly enhanced signal in the ML2-B7 tumor injected with TCR-transduced T _CM whereas the ML2-WT tumors or PBS -injected tumors showed no signal enhancement (Figure 18B and Figure 23B). Quantification of the PET signal revealed a higher percentage of injected dose per gram of tissue (%ID/g) in ML2-B7 tumors injected with TCR2.5D6-transduced T _CM compared to the ML2-WT tumors inoculated with non- transduced T _CM or PBS (Figure 18C). Ex vivo biodistribution showed enhanced uptake in relation to blood in the ML2-B7 tumor compared to ML2-WT tumor, whereas comparable tumor to blood ratios were observed in spleen and lung (Figure 18D). Autoradiography confirmed the data with enhanced radioactivity observed on ML2-B7 tumor sections injected with TCR2.5D6- transduced T _CM (data not shown). Although enhanced signals within the ML2-B7 tumor injected with TCR2.5D6-transgenic T _CM could be detected already at 24h and 12h post injection of ⁸⁹ Zr- aTCRmu-F(ab ^, ) ₂ , quantification of the PET signal, and ex vivo biodistribution showed inferior signal to background ratios at these time points (Figure 23C-D). These data favor 48h post injection as the best time point for imaging TCR-transgenic T _CM by Zr-aTCRmu-F(ab) ₂ .

Investigation of dynamics of Tow-mediated tumor rejection to determine biologically critical imaging time points in a clinically relevant ML2-based myeloid sarcoma model

We optimized the tumor model of myeloid sarcoma by injecting T _CM intravenously in tumor- bearing NSG-mice analog to a potential clinical setting of adoptive T-cell transfer. ML2-B7 and ML2-WT cells were injected subcutaneously in both flanks. Tumor bearing mice were low dose irradiated and TCR-transgenic T _CM , non-transduced T _CM or PBS were transferred intravenously (Figure 24A). Significant rejection of ML2-B7 tumors was observed in mice injected with TCR2.5D6 T _CM in contrast to mice treated with non-transduced T _CM or PBS (Figure 19A). On the other side, ML2-WT tumors showed no modification in growing kinetics following adoptive transfer of TCR-transduced T _CM or controls (Figure 19B). Thus, the established model proved high efficacy of TCR2.5D6-transduced T _CM in a clinically relevant scenario and suggested day 4- 5 after T cell transfer as turning point during rejection and therefore highly interesting for imaging. Ex vivo flow cytometry analyses confirmed a significant increase in total human T cells within the ML2-B7 tumors but not in the ML2-WT tumors (Figure 19C). Human T cells were neither enriched in ML2-B7 nor in ML2-WT tumors in those animals treated with non- transduced T _CM - T-cell infiltration was similar in spleen and lung indicating comparable engraftment ratios (Figure 19C). Around 30% of all T cells within the ML2-B7 tumor were positive for aTCRmu (Figure 19D).

Tracking of TCR2.5D6-transduced 89

T _CM in tumor-bearing NSG mice by ^o:, Zr-aTCRmu- F(ab') ₂ PET imaging in the course of tumor rejection

For monitoring of TCR2.5D6-transduced T _CM in the established in vivo model, we applied Zr- aTCRmu-F(ab') ₂ three days after adoptive T-cell transfer of T _CM or PBS and analyzed mice by PET/CT imaging after 48h (Figure 24B). We detected a highly distinct signal at the ML2-B7 tumor site in animals treated with TCR2.5D6-transduced T _CM whereas no signal was observed on ML2-WT tumors or in animals treated with non-transduced T _CM or PBS (Figure 20A). Quantification of PET signals confirmed these findings (Figure 20B). Ex vivo biodistribution analyses showed a 13-fold increased radioactivity in ML2-B7 tumors in relation to blood in mice injected with TCR2.5D6-transduced T _CM. Furthermore we detected a significant and fourfold increased tumor to blood ratio in ML2-B7 tumors compared to ML2-WT tumors. Mice infused with non-transduced T _CM or PBS showed no differences of tumor to blood ratio between both tumors (Figure 4C). Of note, we detected ex vivo a higher activity in the spleen and lung of mice, which were treated with TCR2.5D6-transduced T _CM (Figure 20C), whereas distribution of ⁸⁹ Zr-aTCRmu-F(ab") ₂ in all other organs was similar independent of the treatment modality (Figure 24C). Autoradiography of tumors and tissue sections confirmed these findings (Figure 20D). Further data validation was performed by ex vivo detection of T cells after imaging. We observed increased numbers of total human T cells in ML2-B7 tumors by flow cytometry, when mice were infused with TCR2.5D6-transduced T _CM (Figure 5A). T-cell infiltration was also detected in ML2-WT tumors, although to a lower extent. In spleen and lung, the percentage of total T cells was comparable for TCR2.5D6-transduced or non-transduced T _CM (Figure 5A). With respect to transgenic T cells, we detected high percentages of TCR2.5D6-transduced T cells within the ML2-B7 tumors but not in the ML2-WT tumors (Figure 2 IB) confirming the specific PET-signal. Slightly enhanced levels of TCR2.5D6-transduced T cells were detected in the lung. Analysis of genomic DNA (gDNA) by qPCR revealed elevated copies of TCR2.5D6 in the ML2-B7 tumor, spleen and lung (Figure 21C) corresponding to biodistribution data. Immunohistochemistry of tumor sections showed tumor cell necrosis and the presence of high numbers of CD5+ T cells within ML2-B7 tumors. In ML2-WT tumors, only few T cells and no signs of tumor necrosis were observed (Figure 2 ID).

Qualitative and quantitative evaluation of ⁸⁹ Zr-aTCRmu-F(ab ^, ) ₂ signals in ML2-B7 tumors

In depth analysis of all mice injected with TCR2.5D6 T _CM (n ⁼ H) revealed differences in distribution of ⁸⁹ Zr-aTCRmu-F(ab") ₂ within ML2-B7 tumors. Based on this signal distribution and the tumor size at the day of imaging, we classified ML2-B7 tumors in three different groups (Figure 22 A). Four mice showed a higher tumor burden and PET signal was detected mostly in the periphery around the ML2-B7 tumor (group I). Tumors of group II (n=4) showed medium tumor size with a high PET signal within the tumor-center. Group III (n=3) demonstrated a small tumor size with reduced signal intensities (Figure 22 A). Although transduction rates of injected TCR2.5D6-transduced T _CM were comparable (66,5% ±3,2%), the tumor volume differed in the groups at the days of T-cell injection (Figure 22B) with larger tumors in group I. Thus, this group represents treatment at a later stage and tumor rejection by transgenic T-cells in an early phase at the day of imaging in comparison to groups II and III (Figure 22B). These observations correlate to the tumor volume calculated by ROI analysis (Figure 25A) and the weight determined ex vivo at the day of imaging Figure 25B). The total %ID in the tumor determined by ROI analysis was enhanced in tumors of group I compared to group II and III (Figure 22C). Analysis of %ID determined ex vivo revealed similar trends Figure 25C). However, %ID/g was found to be the highest for tumors of group II and reduced for group III tumors (Figure 22D). This observation correlates with the observed PET-signal density caused by different distribution patterns or T cell hot spots. Taken together, using the ⁸⁹ Zr-aTCRmu-F(ab') ₂ we classified groups which may reflect the dynamics of T-cell response against a tumor in vivo and resemble the different distribution patterns of T cells within these different rejection phases.

9.3 Discussion

We here present a novel and highly sensitive nuclear imaging approach for tracking of TCR- engineered T cells by directly targeting the transgene using an antibody recognizing the murinized constant TCR beta domain included in the construct as general optimization strategy for transgenic TCR. This approach can be applied for any TCR independent of the defined specificity in case that the distinct murine epitope is present. Herein we used T _CM transduced with TCR2.5D6 specifically recognizing a peptide derived from MPO in a novel myeloid sarcoma model. We demonstrate highly sensitive detection of TCR-transduced effector cells at the tumor site during tumor rejection using the ⁸⁹ Z-aTCRmu-F(ab") ₂ as a specific tracer. We validated our method by ex vivo analyses and observed a significant increase of radioactivity at the matching HLA-B7 tumor in comparison to the non-matching tumor control and directly proved the enrichment of the engineered T cells in the matching tumor.

The usage of the primary transgene for labeling allows omission of additional reporter genes or ex vivo labeling of T cells. The latter are potentially associated to significant disadvantages in respect to reporter gene expression or imaging time (McCracken et al., 2015; Griessinger et al., 2014). However, direct targeting of the TCR complex may have a functional impact on labeled cells and the aTCRmu-IgG has been demonstrated to modulate murine T cell function in vitro and in vivo (Kubo et al., 1989; Drobyski et al., 1996; Nishimura et al., 1994; Kummer et al., 2001). We similarly observed functional modulation of human TCR-transgenic T cells by the aTCRmu-IgG but not by the F(ab') ₂ -fragment.

The number of effector cells infused in our experiments was high but comparable cell numbers were applied in previous clinical trials using genetically modified T cells (Johnson et al., 2009; Robbins et al., 2011; Morgan et al., 2006; Chodon et al., 2014). Previous studies in humans suggested that T-cell engraftment may be more relevant than the absolute number of transferred T cells (Maude et al., 2014). As T _CM engraftment in humans can be expected to be superior as compared to NSG-mice, this preclinical model may even underestimate the potential of this approach in the clinical setting.

We additionally observed different signal distribution patterns within ML2-B7 tumors depending on the tumor size at the time point of imaging. Quantification of the total or weight-adapted signal correlates to qualitative imaging data and ex vivo biodistribution analyses. The comprehensive data analysis may provide information about the stage of tumor rejection and profoundness of tumor cell infiltration by T cells matching to previously published data using microscopic two-photon imaging (Boissonnas et al., 2007). Our data indicate that this noninvasive aTCRmu-F(ab') ₂ -based immuno-imaging technology may reach a sensitivity which may provide deeper insights in pharmacodynamics of transgenic T cells in vivo. The technology may be useful for the timely identification of T cell infiltration versus exclusion from the tumor and therefore for the identification of responders versus non-responders potentially facilitating consecutive modification and optimization of immunotherapeutic approaches.

In addition to the targeting molecule, choice of the nuclear tracer is of particular importance with the need to combine safety and image quality properties. We did not observe an influence of

Zr-labeling on functionality of T cells in vitro. Moreover, T cells effectively destroyed the

on

ML2-B7 tumor in vivo independently of the injection of Zr-aTCRmu-F(ab') ₂ .

Specific signals detected by PET/CT were intensively validated ex vivo by biodistribution and autoradiography as well as the detection of TCR-engineered T _CM by flow cytometry and qPCR. These analyses confirmed the excellent signal-to-noise relation provided by PET/CT. We detected enhanced radioactivity in spleen and lung in animals treated with TCR-transduced T _CM corresponding to higher amounts of TCRmu-positive T cells. Within the spleen we observed high amounts of T cells, but the percentage of transgenic CD5+TCRmu+ cells was comparably low, confirming the spleen as organ in which human T cells easily engraft (27). The percentage of CD5+TCRnui+ cells was relatively high within the lung although only limited numbers of total human T cells were detected. This may be explained by innate signals inducing T cell recruitment and is not necessarily associated to specific T cell functions (Galkina et al, 2005).

The features of the present invention disclosed in the specification, the claims, and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof. Tables

Table 1. Identified HLA ligands derived from genes with restricted expression to the hematopoietic system

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