EP4665752A1 - Il-15 fusion proteins with improved properties - Google Patents

Abstract

The present invention relates to a fusion protein comprising (i) a binding protein that specifically binds to a cell-surface antigen on a target cell of interest, and (ii) a modified IL-15 protein wherein said modified IL-15 protein has an amino acid sequence being at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1 and wherein said modified IL-15 protein has an amino acid substitution of L to E at a position corresponding to position 45 of SEQ ID NO: 1, an amino acid substitution of N to E at a position corresponding to position 72 of SEQ ID NO: 1, an amino acid substitution of E to Q at a position corresponding to position 93 of SEQ ID NO: 1; and/or an amino acid substitution of F to L at a position corresponding to position 103 of SEQ ID NO: 1. The present invention further contemplates a polynucleotide encoding the fusion protein, a vector or expression construct comprising the polynucleotide, a host cell comprising the polynucleotide, the vector or expression construct or a non-human transgenic multicellular organism comprising the polynucleotide, vector or expression construct or the host cell. The present invention also relates to a method for the manufacture of a fusion protein and to a medicament comprising the fusion protein, the polynucleotide, the vector or expression construct or the host cell. Yet, the said fusion protein, the polynucleotide, the vector or expression construct or the host cell are provided for use in treating and/or preventing a disease or medical condition in a subject.

Description

IL-15 fusion proteins with improved properties

The present invention relates to a fusion protein comprising (i) a binding protein that specifically binds to a cell-surface antigen on a target cell of interest, and (ii) a modified IL- 15 protein wherein said modified IL- 15 protein has an amino acid sequence being at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1 and wherein said modified IL- 15 protein has an amino acid substitution of L to E at a position corresponding to position 45 of SEQ ID NO: 1, an amino acid substitution of N to E at a position corresponding to position 72 of SEQ ID NO: 1, an amino acid substitution of E to Q at a position corresponding to position 93 of SEQ ID NO: 1; and/or an amino acid substitution of F to L at a position corresponding to position 103 of SEQ ID NO: 1. The present invention further contemplates a polynucleotide encoding the fusion protein, a vector or expression construct comprising the polynucleotide, a host cell comprising the polynucleotide, the vector or expression construct or a non-human transgenic multicellular organism comprising the polynucleotide, vector or expression construct or the host cell. The present invention also relates to a method for the manufacture of a fusion protein and to a medicament comprising the fusion protein, the polynucleotide, the vector or expression construct or the host cell. Yet, the said fusion protein, the polynucleotide, the vector or expression construct or the host cell are provided for use in treating and/or preventing a disease or medical condition in a subject.

Second generation chimeric or humanized monoclonal antibodies (mAbs) directed to tumor associated antigens (TAAs) are a cornerstone of oncological treatment. Rituximab was the first antitumor mAb to become clinically available and has considerably improved treatment outcome in patients with B cell lymphoma. Its therapeutic efficacy is largely mediated by its capability to recruit Fc-receptor (FcR)-positive immune effector cells, in particular NK cells. In general, however, the therapeutic activity of such antitumor antibodies is limited and there remains an urgent medical need for development of optimized reagents.

The capability to recruit Fc-receptor (FcR)-positive immune effector cells, such as NK cells, is considered as being crucial for the therapeutic activity of most anti-tumor antibodies. Thus, many of the strategies used for antibody optimization focus on the improvement of the Fc-part resulting in an enhanced antibody dependent cellular cytotoxicity (ADCC)-activity. For instance, various approaches aim to enhance affinity to the Fc-receptor Illa (FcRIIIa/CD16a) by genetic engineering of the glycosylation pattern and/or the amino acid sequence of the CH2 domain of the IgGl-Fc part contained in most antitumor mAbs (Lazar et al, 2006; Shinkawa et al, 2003). Roche in cooperation with Glycart, for example, has developed the glycoengineered CD20- mAb GAI 01 (Obinutuzumab) (Umana et al, 2007), whereas Xencor (Monrovia, CA, USA) developed an mAb directed to CD19 (XmAb5574/MOR208, Tafasitamab) that carries the amino acid exchanges S239D and I332E (SDIE-modification) (Dull et al, 2021; Horton et al, 2008) that are approved for treatment of chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma, respectively (Dull et al, 2021; Lee et al, 2014). As GA101, these antibodies were reported to exert markedly ADCC and are currently evaluated in clinical trials. Despite these improvements, therapeutic activity of these and other antitumor mAbs is still not satisfactory (Beck et al, 2010).

Another approach to improve efficacy conceptualized already in the early nineties is to fuse mAbs to cytokines with known antitumor activity. In this regard, IL-2 and IL- 15 appear particularly promising, as they stimulate antitumor immunity not only by reinforcing activation, but also by inducing effective proliferation of NK cells (Sun et al, 2019), the latter being a critical prerequisite to combat higher tumor burden. Whereas both IL-2 and IL- 15 stimulate NK and effector T cells (Waldmann, 2006), they differ regarding their effects on regulatory T cells (Treg). While it is certain that IL-2 causes undesired activation of Treg, data on IL- 15 are considered controversial (Ben Ahmed et al, 2009; Clark & Kupper, 2007; Imamichi et al, 2008; Perna et al, 2013; Raynor et al, 2013; Vang et al, 2008; Waldmann, 2006; Waldmann, 2015; Xia et al, 2010), but it is generally believed that IL- 15 compares favorably to IL-2 with respect to the attenuated activation of T reg cells. In addition, available data clearly indicate that IL- 15 is superior for induction of anti-tumor immunity. This is supported by impressive effects of IL- 15 against large, established tumors in mice (Liu et al, 2013). Immunocytokines using IL- 15 variants have been provided and are described in, e.g., EP 3 265 478 A.

However, many of these proteins suffer from aggregation to a variable degree. The formation of aggregates hampers not only production, and thus, the development of fusion proteins as a therapeutic reagent, but possibly also biological function.

Thus, there is a need for fusion proteins with increased solubility and production rates.

The technical problem underlying the present invention may be seen as the provision of means and methods for complying with the aforementioned need. The technical problem is solved by the embodiments characterized in the claims and herein below. The invention, thus, relates to a fusion protein comprising:

(i) a binding protein that specifically binds to a cell-surface antigen on a target cell of interest, and

(ii) a modified IL- 15 protein wherein said modified IL- 15 protein has an amino acid sequence being at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1 and wherein said modified IL- 15 protein has an amino acid substitution of L to E at a position corresponding to position 45 of SEQ ID NO: 1, an amino acid substitution of N to E at a position corresponding to position 72 of SEQ ID NO: 1, an amino acid substitution of E to Q at a position corresponding to position 93 of SEQ ID NO: 1; and/or an amino acid substitution of F to L at a position corresponding to position 103 of SEQ ID NO: 1

It is to be understood that in the specification and in the claims, “a” or “an” can mean one or more of the items referred to in the following depending upon the context in which it is used. Thus, for example, reference to “an” item can mean that at least one item can be utilized.

As used in the following, the terms “have”, “comprise” or “include” are meant to have a nonlimiting meaning or a limiting meaning. Thus, having a limiting meaning these terms may refer to a situation in which, besides the feature introduced by these terms, no other features are present in an embodiment described, i.e. the terms have a limiting meaning in the sense of “consisting of’ or “essentially consisting of’. Having a non-limiting meaning, the terms refer to a situation where besides the feature introduced by these terms, one or more other features are present in an embodiment described.

Further, as used in the following, the terms “preferably”, “more preferably”, “most preferably”, "particularly", "more particularly", “typically”, and “more typically” are used in conjunction with features in order to indicate that these features are preferred features. However, the terms shall indicate that alternative features may also be envisaged in accordance with the invention.

Further, it will be understood that the term “at least one” as used herein means that one or more of the items referred to following the term may be used in accordance with the invention. For example, if the term indicates that at least one item shall be used this may be understood as one item or more than one item, i.e. two, three, four, five or any other number. Depending on the item the term refers to the skilled person understands as to what upper limit the term may refer, if any.

The term "about" in the context of the present invention means +/- 20%, +/- 10%, +/- 5%, +/- 2 % or +/- 1% from the indicated parameters or values. This also takes into account usual deviations caused by measurement techniques and the like.

The term “fusion protein” as used herein refers to a protein comprising two or more peptide or protein units linked to each other by peptide bonds. Accordingly, a fusion protein of the invention shall at least comprise the entire or a partial amino acid sequence from a first protein or peptide and the entire or a partial amino acid sequence from a second protein or peptide. The protein or peptide units of the fusion protein are linked to each other via peptide bonds. The amino acid sequence of the first protein or peptide unit may be directly bound on its C-terminal end to the N-terminal end of the amino acid sequence of the second protein or peptide or vice versa. Alternatively, there might be a linker region introduced between the amino acid sequence of the first and the second protein or peptide unit. Such a linker region may, preferably, comprise or essentially consist of between about 1 and about 20 amino acids, more preferably, between about 1 and about 15 amino acids, about 1 and about 10 amino acids, about 1 and about 8 amino acids, about 1 and about 7 amino acids, about 1 and about 6 amino acids or about 1 and about 5 amino acids. Accordingly, the fusion protein can be encoded by a single polynucleotide and will be synthesized as a single fusion protein molecule. It will be understood that there may be posttranslational rearrangements due to proteolytic cleavage or other posttranslational modifications that may take place on the fusion protein. Yet, the fusion protein in accordance with the present invention may also comprise the entire or a partial amino acid sequence of a third, fourth, fifth protein or peptide and so on.

The fusion protein according to the present invention shall comprise at least two protein or peptide units, i.e. (i) a binding protein that specifically binds to a cell-surface antigen on a target cell of interest, and (ii) a modified IL-15 protein as specified elsewhere herein in more detail. Preferably, the fusion protein of the invention, however, essentially consists of those two units i.e. lacks further protein or peptide units.

The term “binding protein” refers to any protein capable of binding to a cell surface antigen on the surface of a target cell of interest, e.g., a tumor specific protein or a protein expressed on the surface of a circulating tumor cell. Typical binding proteins may be antibodies or fragments thereof, receptor proteins or other proteins that allow for specific binding of a target structure of interest, such as, lipocalin-based binding proteins, abtyrin-based binding proteins, crystalline-based binding proteins, adnectins, EGF-like domain proteins, Kringel-domain proteins, fibronectin type 1 domain proteins, fibronectin type II domain proteins, fibronectin type III domain proteins, PAN domain, proteins G1 a domain proteins, SRCR domain proteins, Kunitz/Bovine pancreatic trypsin Inhibitor domain proteins, Kazal-type serine protease inhibitor domain proteins, Trefoil (P-type) domain proteins, von Willebrand factor type C domain proteins, Anaphylatoxin-like domain proteins, CUB domain proteins, thyroglobulin type 1 repeat proteins, LDL-receptor class A domain proteins, Link domain proteins, Thrombospondin type 1 domain proteins, C-type lectin domain proteins, MAM domain proteins, von Willebrand factor type A domain proteins, Somatomedin B domain proteins, WAP -type four disulfide core domain proteins, F5/8 type C domain proteins, Hemopexin domain proteins, SH2 domain proteins, SH3 domain proteins, Laminin- type EGF-like domain proteins, ubiquitins, zinc-finger proteins, or leucine-rich repeat proteins.

Preferably, the binding protein is an antibody or antibody-binding fragment thereof.

The term “antibody” as used herein refers to a full-length antibody, a recombinant antibody molecule, or a fully human antibody molecule. A full-length antibody is any naturally occurring antibody. The term "antibody" also includes immunoglobulins (Ig's) of different classes (i.e. IA, IgG, IgM, IgD and IgE) and subclasses (such as IgGl, IgG2 etc.). Such full-length antibodies can be isolated from different animals such as e.g. different mammalian species. A recombinant antibody molecule refers to an antibody molecule the genes of which has been cloned, and that is produced recombinantly in a host cell or organism, using well-known methodologies of genetic engineering. Typically, a recombinant antibody molecule has been genetically altered to comprise an amino acid sequence, which is not found in nature. Thus, a recombinant antibody molecule can be a chimeric antibody molecule or a humanized antibody molecule. The binding protein of the fusion protein of the present invention can also be an "antibody-binding fragment". Such antibody-binding fragments comprise at least those parts of an antibody, that form the (antigen) binding site. Preferably, such antibody-binding fragments are single chain variable fragments (scFv), Fv fragments, single domain antibodies, such as e.g. VHH (camelid) antibodies, di-scFvs, fragment antigen binding regions (Fab), F(ab')2 fragments, Fab' fragments, diabodies, domain antibodies, or bispecific "Fabsc"-antibody molecules as described in International patent application W02013/092001 comprising a single chain Fv fragment which is connected to an Fab fragment via a CH2 domain to name only a few. Preferred divalent antibody fragments include a (Fab)2'-fragment, abispecific single-chain Fv fragment, a bsFc-l/2-dimer or a bsFcCH3-l/2 dimer as described in International Patent Application W02013/092001. Binding protein of the fusion protein of the present invention may however only have a single binding site, i.e., may be monovalent. Preferably, monovalent binding proteins include a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties. More preferably, such monovalent antibody fragments include an Fab fragment, an Fv fragment, a single-chain Fv fragment (scFv) or an scFv-Fc fragment.

An antibody or fragment thereof used as a binding protein in accordance with the fusion protein of the invention may comprise modifications that enhance or attenuate Fc activity of the antibody such as binding of the said antibody or fragment thereof by Fc receptors. In particular, an antibody as binding protein of the fusion protein of the present invention may be modified such that it has an enhanced antibody dependent cellular toxicity (ADCC) activity compared to an unmodified binding protein. The ADCC activity can be measured by well-known assays, such as e.g. CellaTM-TOX assay, GAPDH release assay, which can be obtained from e.g. Promega or Interchim. Thus, an antibody as binding protein of the fusion protein of the present invention may have an increased ADCC activity when compared to the same but unmodified antibody. Preferably, a modification of the antibody is present in the Fc part of the antibody or antibody-binding fragment thereof. Even more preferably, the modification is an SDIE mutation in the Fc part of the antibody or antibody-binding fragment thereof. SDIE mutations are known to mediate markedly enhanced affinity to Fc receptors as well as ADCC (e.g. Lazar et al., 2006; Horton et al. (2008); Foyil and Bartlett, 2010). Specifically, the SDIE mutation refers to an amino acid substitution comprising S239D and I332E, wherein the positional numbering is according to the EU index. It is also envisioned that the indicated amino acid substitutions correspond to the indicated amino acid positions. That means that, for example, in antibody fragments or binding proteins comprising an Fe domain the positional numbering of the indicated amino acids may differ but may still have similar neighboring amino acids as also described herein below in more detail.

As used herein, the term “specifically binds” is understood to mean that the binding protein has a higher (selective) affinity for a particular cell-surface antigen or a region or portion of the cell-surface antigen on a target cell. Due to said selective affinity, the binding protein does preferably not cross-react, i.e. bind, to other cell-surface antigens. Specificity may be tested by binding assays known in the art including Western blot assays, immunoassay such as ELISA, FACS analysis, biacore assays, surface plasmon resonance assays and the like. Specific binding as referred to herein occurs with a specific dissociation constant (KD). The dissociation constant may be, preferably, about 1 pM (10⁶ M) or less, more preferably about 100 nm (10⁷ M) or less, more preferably about 10 nM (10⁸ M) or less, more preferably about 1 nM (10⁹ M) or less, more preferably about 100 pM (10¹⁰ M) or less, or more preferably about 10 pM (10¹¹ M) or less. A high affinity corresponds to a low value of KD. Appropriate controls as known in the art can be used to distinguish between “specific” and “non-specific” binding. Specific binding means herein that a binding protein binds stronger to a cell-surface antigen, such as an epitope for which it is specific, compared to the binding to another molecule. Preferably, the dissociation constant for the cell-surface antigen to which the binding protein binds specifically is more than about 10-fold, preferably more than about 20-fold, more preferably more than about 50-fold, even more preferably more than about 100-fold, about 200-fold, about 500-fold, or about 1000- fold lower than the dissociation constant for the molecule to which the binding protein does not bind specifically.

The term “dissociation constant” or “KD” defines the specific binding affinity. As used herein, the term "KD" (usually measured in "mol/1", sometimes abbreviated as "M") is intended to refer to the dissociation equilibrium constant of the particular interaction between a first compound and a second compound. As referred to herein, the term KD is particularly used to describe the binding affinity between the fusion protein, more specifically the binding protein of a fusion protein, and a target protein, i.e. a cell-surface antigen on atarget cell. The KD can be determined by ELISA or by surface plasmon resonance (SPR) assays.

The term “cell-surface antigen” as referred to herein relates to a molecule that is exposed at the cell surface and that can be recognized and bound by the binding molecule. Typically, a cellsurface antigen as meant herein is a protein exposed at the cell surface. Such a protein may be a transmembrane protein exhibiting an extracellular portion at the cell surface or a cell membrane-associated extracellular protein. Yet, a cell-surface antigen may also be a non- proteinaceous molecule such as a lipid or sugar exhibited on the cell surface. Preferably, the cell-surface antigen envisaged in accordance with the present invention is specific for the target cell of interest, i.e. it is present exclusively or pivotally on said target cells such that the binding agent can specifically direct the fusion protein to said target cells.

Preferred cell-surface antigens envisaged in accordance with the present invention are antigens associated with tumors (TAAs) and/or antigens associated with autoimmune diseases.

TAAs can be, preferably, selected from the group consisting of CD 19, CD20, CD 10, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD38, CD44v6, CD45, CDw52, Fms-like tyrosine kinase 3 (FL T-3, CD135), c-Kit (CD117), CSF1 R, (CD115), CD123, CD133, PDGFR-a (CD140a), PDGFR-P (CD140b), chondroitin sulfate proteoglycan 4 (CSPG4, melanoma- associated chondroitin sulfate proteoglycan), Muc-1, EGFR, de2-7-EGFR, EGFRylll, Folate blocking protein, Her2neu, Her3, PSMA, PSCA, PSA, TAG-72, HLA-DR, IGFR, CD133, IL3R, fibroblast activating protein (FAP), Carboanhydrase IX (MN/CA IX), Carcinoembryonic antigen (CEA), EpCAM, CDCP1, Derlinl, Tenascin, frizzled 1-10, the vascular antigens VEGFR2 (KDR/FLK1 ), VEGFR3 (FL T4, CD309), Endoglin, CLEC14, Teml-8, Tie2, mesothelin, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), cancer antigen 72-4 (CA72-4 ), interleukin 13 receptor alpha-2 subunit, IL 13Ra2, Ig kappa light chain (K), GD3 -ganglioside (GD3), GD2-ganglioside (GD2), acetylated variants of GD2 and GD3, CD171, NCAM, alpha folate receptor (aFR), Lewis(Y), fetal acetylcholine receptor (FAR), avian erythroblastic leukemia viral oncogene homolog 3 (ERBB3), avian erythroblastic leukemia viral oncogene homolog 4 (ERBB4 ), avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2), hepatocyte growth factor receptor (HGFR/c-Met), claudin 18.2, claudin 3, claudin 4, claudin 1, claudin 12, claudin 2, claudin 5, claudin 8, claudin 7, claudin 6, membrane bound CEA, Robo4 and CD 138, B7-H3 (CD276), BCMA, GRPC5D,.

Antigens associated with autoimmune disease include, preferably, CD20, CD22, CD52, TNFR, CD19, CD25 PD1, PDL1 and CD40 and CD47.

The term “target cell of interest” refers to any cell capable of expressing at least one of the cellsurface antigens as referred to herein. Preferably, the target cell of interest is a cancer cell or a cell involved or associated with an autoimmune disease.

Preferably, said binding protein that specifically binds to a cell-surface antigen on a target cell of interest is an antibody or antibody fragment. More preferably, said antibody or antibody fragment is an anti-CD19 antibody or antibody fragment.

The term “IL- 15” as used herein refers to interleukin 15 which is a cytokine with various biological activities reported in the prior art including activation and stimulation of the proliferation of T cells, and, in particular, memory CD8⁺ cells, as well as NK cells. IL- 15 binds under physiological conditions to a specific IL- 15 receptor alpha (IL-15Ra) and to the IL-2/IL- 15 receptor beta gamma (fL-2/IL-15RPy). The latter receptor is, typically, bound and activated by both, IL-2 and IL-15. In contrast to IL-2, IL- 15 triggers its receptor in trans, that is the IL- 15Ra expressed on monocytes and dendritic cells trans-stimulates the Py-receptor on NK- and T cells. IL- 15 proteins are known from various species including humans. A human IL- 15 protein as referred to herein, preferably, has an amino acid sequence as shown in SEQ ID NO: 1. It will be understood that IL- 15 is a secreted protein. Accordingly, it is synthesized within the cell containing a signal sequence, which is cleaved-off from the molecule during maturation and secretion. For human IL-15, different isoforms are reported that differ in the length of their signal peptides. The aforementioned SEQ ID NO: 1 shows the amino acid sequence of the secreted IL- 15 protein. The term “modified IL- 15 protein” refers to an IL- 15 protein, which compared to wildtype IL- 15 comprises at least one amino acid substitution, addition and/or deletion. Wildtype IL-15 as referred to herein, preferably, is an IL- 15 protein having an amino acid sequence as shown in SEQ ID NO : 1. However, it will be understood that due to allelic variations within different subjects of a species or variations between different species, wildtype IL- 15 may also encompass IL- 15 proteins having a sequence that differs from SEQ ID NO: 1 as long as those IL- 15 proteins exhibit essentially the same biological and immunological properties as IL- 15 having SEQ ID NO: 1 and have an amino acid sequence being at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1. Moreover, there are IL- 15 protein variants described that differ from wildtype IL- 15 in one or more amino acids which have altered receptor binding activities. Some IL- 15 variants have decreased binding affinity for IL-15Ra while others have increased binding activity for said receptor. Similarly, there are reports for amino acid substitutions that influence IL-2/IL-15RPy binding (see W02005/085282 or EP 3 265 478 A). Those IL-15 variants are also encompassed as modified IL-15 proteins as long as they have at least one of the above mentioned specific amino acid substitutions, have an amino acid sequence being at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1 and if present in the fusion protein according to the invention are capable of eliciting enhanced NK cell proliferation and/or anti-tumor activity compared to a control fusion protein, reducing aggregation compared to a control fusion protein and/or exhibiting increased production rates compared to a control fusion protein in host cells. A modified IL- 15 protein in accordance with the present invention shall have an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence identified by a SEQ ID NO: 1.

Sequence identity between two amino acid sequences as referred to herein, in general, can be determined by alignment of two sequences either over the entire length of one of the sequences or within a comparison window. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment and calculation of sequence identity can be done by using published techniques or methods codified in computer programs such as, for example, BLASTP, BLASTN or FASTA. The percent sequence identity values are, preferably, calculated over the entire amino acid sequence. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To carry out the sequence alignments, the program PileUp or the programs Gap and BestFit, which are part of the GCG software packet (Genetics Computer Group, US), may be used. The sequence identity values recited above in percent (%) are to be determined, in another aspect of the invention, using the program GAP over the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which, unless otherwise specified, shall always be used as standard settings for sequence alignments.

The modified IL- 15 protein according to the present invention, furthermore, shall at least contain one or more of the following amino acid substitutions:

• an amino acid substitution of L to E at a position corresponding to position 45 of SEQ ID NO: 1,

• an amino acid substitution of N to E at a position corresponding to position 72 of SEQ ID NO: 1,

• an amino acid substitution of E to Q at a position corresponding to position 93 of SEQ ID NO: 1;

• and/or

• an amino acid substitution of F to L at a position corresponding to position 103 of SEQ ID NO: 1.

The term “amino acid position” according to the present invention means the position number of an amino acid within an amino acid sequence as shown herein. Thus, an a given amino acid, e.g., at position 45 means that this amino acid is at the position number 45 when counting from the first N-terminal amino acid in a given amino acid sequence. The term “corresponding” means that a position is not in each molecule determined by the number of the preceding amino acids, i.e. its position number. The position number of a give amino acid in a variant amino acid sequence in accordance with the present invention could vary due to deleted or additional amino acids prior to the said position number compared to a reference amino acid sequence. Thus, under the term “corresponding to position” it is to be understood that amino acids may differ in the indicated position number but may still have similar neighboring amino acids and may fulfill the same structural or biological functions in the molecule as the corresponding amino acid does ion the reference.

Particular preferred modified IL- 15 proteins are those having an amino acid sequence as shown in SEQ ID NO: 2 (L45E variant), 3 (N72E variant), 4 (E93Q variant) or 5 (F103L variant).

Preferably, said modified IL- 15 protein of the fusion protein of the invention has a reduced affinity for the IL- 15 receptor alpha compared to a wildtype IL- 15 protein. More preferably, said modified IL- 15 protein of the fusion protein is capable of binding to IL-2/IL-15 receptor beta/gamma with essentially the same affinity or higher affinity as wildtype IL- 15 protein. The term “reduced affinity” as used herein relates to an affinity, which is reduced to a statistically significant degree. Preferably, the reduced affinity results in a reduction of the association rate, an increase in the dissociation rate or a decreased time of binding between the modified IL- 15 protein and its IL- 15 receptor alpha. More preferably, reduced affinity as referred to herein means that no apparent binding of the modified IL- 15 protein to the IL- 15 receptor alpha takes place at all.

More preferably, the said modified IL-15 protein has an amino acid substitution of E to K at a position corresponding to position 46 of SEQ ID NO: 1. The E46K amino acid substitution in IL- 15 proteins was found to abolish effectively binding to the IL- 15 receptor alpha and, thus, undesired off-target effects in vivo. Nevertheless, other amino acid substitutions or combinations thereof may have similar effects. Suitable substitutions are also described in W02005/085282 or EP 3 265 478 A.

In a preferred embodiment of the fusion protein of the invention, the said fusion protein is capable of eliciting enhanced NK cell proliferation and/or anti-tumor activity compared to a control fusion protein. Preferably, said fusion protein comprises a modified IL- 15 protein wherein said modified IL- 15 protein has an amino acid sequence being at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1 and wherein said modified IL- 15 protein has an amino acid substitution of L to E at a position corresponding to position 45 of SEQ ID NO: 1, an amino acid substitution of N to E at a position corresponding to position 72 of SEQ ID NO: 1; and/or an amino acid substitution of E to Q at a position corresponding to position 93 of SEQ ID NO: 1.

In yet a preferred embodiment of the fusion protein of the invention, the said fusion protein exhibits reduced aggregation compared to a control fusion protein. Preferably, said fusion protein comprises a modified IL- 15 protein wherein said modified IL- 15 protein has an amino acid sequence being at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1 and wherein said modified IL- 15 protein has an amino acid substitution of L to E at a position corresponding to position 45 of SEQ ID NO: 1, and/or an amino acid substitution of F to L at a position corresponding to position 103 of SEQ ID NO: 1. Also, in a preferred embodiment of the fusion protein of the invention, the said fusion protein exhibits increased production rates compared to a control fusion protein in host cells, preferably, CHO cells. Preferably, said fusion protein comprises a modified IL- 15 protein wherein said modified IL- 15 protein has an amino acid sequence being at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1 and wherein said modified IL- 15 protein has an amino acid substitution of L to E at a position corresponding to position 45 of SEQ ID NO: 1, an amino acid substitution of E to Q at a position corresponding to position 93 of SEQ ID NO: 1; and/or an amino acid substitution of F to L at a position corresponding to position 103 of SEQ ID NO: 1.

The term “control fusion protein” refers to a fusion protein that comprises a binding protein as specified above and an unmodified IL- 15 protein. The control fusion protein may contain wildtype IL- 15 protein or a variant thereof as specified elsewhere herein. However, the term “unmodified IL- 15 protein” refers to an IL- 15 protein that lacks any one of the above mentioned amino acid substitutions L45E, N72E, E93Q and F103L. Thus, preferably, said unmodified IL- 15 protein is an IL- 15 protein as specified above except that it lacks any of the amino substitutions L45E, N72E, E93Q and F103L. It will be understood that the control fusion protein as referred to herein may also have a reduced affinity for the IL- 15 receptor alpha compared to a wildtype IL- 15 protein and/or may still be capable of binding to IL-2/IL-15 receptor beta/gamma with essentially the same affinity or higher affinity as wildtype IL- 15 protein. More preferably, the control fusion protein of the present invention may also comprise the E46K amino acid substitution described elsewhere herein.

Preferably, the fusion protein of the present invention has an amino acid sequence as shown in any one of SEQ ID NOs: 6, 7, 8 or 9.

Advantageously, it has been found in the studies underlying the present invention that a fusion protein according to the present invention exhibits improved folding and accordingly superior production rate as well as significantly lower proportion of aggregates when expressed and produced in host cells. In addition, the fusion protein of the present invention showed improved NK cell proliferation and activation of IL-15Py. Moreover, it has been found that the fusion constructs exhibited potent anti-tumor activity both in vitro and in vivo. Thanks to the present invention, IL- 15 immunocytokines that are particular biologically effective in eliciting enhanced NK cell proliferation and/or anti-tumor activity can be more efficiently produced.

The explanations and definitions of the terms made above apply mutatis mutandis for the embodiments of the invention described herein below.

The invention also relates to a polynucleotide encoding the fusion protein of the invention.

The term “polynucleotide” as used in accordance with the present invention refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). The term as used herein encompasses the sequence specified herein as well as the complementary or reverse- complementary sequence thereof. Preferably, the polynucleotide is RNA or DNA. The term also encompasses DNAs or RNAs with backbones modified for stability or for other reasons. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are also encompassed as polynucleotides. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. Every nucleic acid sequence herein that encodes a certain polypeptide of the invention may, due to the degeneracy of the genetic code, have silent variations. The degeneracy of the genetic code yields a large number of functionally identical polynucleotides that encode the same polypeptide. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are silent variations.

The polynucleotide of the invention shall encode the fusion protein of the invention, i.e. it shall comprise a nucleic acid sequences, which encodes said fusion protein of the invention. In addition, the polynucleotide of the present invention may comprise additional nucleic acid sequences. Preferably, the polynucleotide of the present invention may comprise in addition to an open reading frame further untranslated sequence at the 3’ and at the 5’ terminus of the coding gene region: at least 500, preferably 200, more preferably 100 nucleotides of the sequence upstream of the 5’ terminus of the coding region and at least 100, preferably 50, more preferably 20 nucleotides of the sequence downstream of the 3’ terminus of the coding gene region.

The polynucleotide of the present invention shall be provided, preferably, either as an isolated polynucleotide (i.e. purified or at least isolated from its natural context such as its natural gene locus) or in genetically modified or exogenously (i.e. artificially) manipulated form. An isolated polynucleotide can, for example, comprise less than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences, which naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. The polynucleotide, preferably, is provided in the form of double or single stranded molecule. It will be understood that the present invention by referring to any of the aforementioned polynucleotides of the invention also refers to complementary or reverse complementary strands of the specific sequences or variants there-of referred to before. The polynucleotide encompasses DNA, including cDNA and genomic DNA, or RNA polynucleotides.

Moreover, comprised are also chemically modified polynucleotides including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides or artificial modified ones such as biotinylated polynucleotides.

Preferably, the polynucleotide of the present invention has a nucleotide sequence as shown in SEQ ID NO: 10. However, it will be understood that a polynucleotide of the present invention may also have a variant nucleotide sequence that comprise at least one nucleotide deletion, addition and/or substitution compared to the nucleotide sequences shown in the aforementioned SEQ ID NO. Such a variant polynucleotide, however, shall still encode a fusion protein having the biological functions and properties referred to above. The polynucleotide of the present invention may, thus, have an nucleotide sequence being at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% or at least about 99% identical to the nucleotide sequence shown in SEQ ID NO: 10. Nucleotide sequence identity can be determined, preferably, by methods well known in the art and referred to elsewhere herein. More typically, nucleotide sequence identity may be determined by aligning two nucleic acid sequence such that the highest degree of matches is achieved. This can be done by using well known algorithms of Altschul or Needelman & Wunsch or Smith & Waterman or those implemented in the BLASTN, FASTA, GCG, GAP, BestFit or Pileup programs. An alignment of two nucleic acid sequences may be a global alignment, i.e. an alignment over the entire range of the nucleotide sequences to be compared or a local alignment, i.e. an alignment over a stretch nucleotides of significant length in both sequences. Typically, the standard settings of the aforementioned programs shall be used for carrying out comparison. Moreover, the polynucleotide of the present invention may also be a polynucleotide encoding a fusion protein having the biological functions and properties referred to above that is capable of hybridizing under stringent hybridization conditions to the polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 10. Stringent hybridization conditions as referred to in accordance with the present invention are described in standard text books such as Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and well known to the skilled artisan. Preferably, stringent hybridization conditions are hybridization in 6 x sodium chloride/sodium citrate (SSC) at approximately 45°C, followed by one or more wash steps in 0.2 x SSC, 0.1% SDS at 50 to 65°C. The skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example, when organic solvents are present, with regard to the temperature and concentration of the buffer.

Yet, the present invention relates to a vector or expression construct comprising the polynucleotide of the invention.

The term “vector”, preferably, encompasses phage, plasmid, cosmids, viral vectors as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes (YAC). The vector encompassing the polynucleotide of the present invention, preferably, further comprises selectable markers for propagation and/or selection in a host. The vector may be incorporated into a host cell by various techniques well known in the art. If introduced into a host cell, the vector may reside in the cytoplasm or may be incorporated into the genome. In the latter case, it is to be understood that the vector may further comprise nucleic acid sequences, which allow for homologous recombination or heterologous insertion. Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection”, conjugation and transduction, as used in the present context, are intended to comprise a multiplicity of prior-art processes for introducing foreign nucleic acid (for example DNA) into a host cell, including calcium phosphate, rubidium chloride or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, f-mating, natural competence, carbon-based clusters, chemically mediated transfer, electroporation or particle bombardment. Suitable methods for the transformation or transfection of host cells, including plant cells, can be found in standard textbooks such as Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Alternatively, a plasmid vector may be introduced by heat shock or electroporation techniques. Should the vector be a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells. Preferably, the vector of the present invention is an expression vector. In such an expression vector, i.e. a vector which comprises the polynucleotide of the invention having the nucleic acid sequence operatively linked to an expression control sequence (also called “expression cassette”) allowing expression in prokaryotic or eukaryotic cells or isolated fractions thereof. Suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDVl (Pharmacia), pCDM8, pRc/CMV, pcDNAl, pcDNA3 (Invitrogene) or pSPORTl (GIBCO BRL). Further examples of typical fusion expression vectors are pGEX, pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), where glutathione S transferase (GST), maltose E-binding protein and protein A, respectively, are fused with the recombinant target protein. Examples of suitable inducible non-fusion E. coli expression vectors are, inter alia, pTrc and pET l id. The tar-get gene expression of the pTrc vector is based on the transcription from a hybrid trp-lac fusion promoter by host RNA polymerase. The target gene expression from the pET l id vector is based on the transcription of a T7-gnl0-lac fusion promoter, which is mediated by a co-expressed viral RNA polymerase (T7 gnl). This viral polymerase is provided by the host strains BL21 (DE3) or HMS174 (DE3) from a resident lambda-prophage, which harbors a T7 gnl gene under the transcriptional control of the lacUV 5 promoter. The skilled worker is familiar with other vectors, which are suitable in prokaryotic organisms; these vectors are, for example, in E. coli, pLG338, pACYC184, the pBR series such as pBR322, the pUC series such as pUC18 or pUC19, the Ml Bmp series, pKC30, pRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-IIIl 13-B1, lambdagtl l or pBdCl, in Streptomyces plJlOl, plJ364, plJ702 or plJ361, in Bacillus pUBUO, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667. Examples of vectors for expression in the yeast S. cerevisiae comprise pYep Seel, pMFa, pJRY88 and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and processes for the construction of vectors, which are suitable for use in other fungi, such as the filamentous fungi, comprise those, which are described in detail in standard textbooks, such as van den Hondel, C.A.M.J.J., & Punt, P.J. (1991) “Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, J.F. Peberdy et al., Ed., pp. 1-28, Cambridge University Press: Cambridge, or in: More Gene Manipulations in Fungi (J.W. Bennett & L.L. Lasure, Ed., pp. 396-428: Academic Press: San Diego). Further suitable yeast vectors are, for example, pAG-1, YEp6, YEpl3 or pEMBLYe23. As an alternative, the polynucleotides of the present invention can be also expressed in insect cells using baculovirus expression vectors. Baculovirus vectors, which are available for the expression of proteins in cultured insect cells, e.g., Sf9 cells, comprise the pAc series and the pVL series.

Yet the vector may be an integration vector. An integration vector refers to a DNA molecule, linear or circular, that can be incorporated, e.g., into a microorganism's genome, such as a bacteria’s genome, and provides for stable inheritance of a gene encoding a polypeptide of interest, such as the alcohol acyl transferase of the invention. The integration vector generally comprises one or more segments comprising a gene sequence encoding a polypeptide of interest under the control of additional nucleic acid segments that provide for its transcription.

Such additional segments may include promoter and terminator sequences, and one or more segments that drive the incorporation of the gene of interest into the genome of the target cell, usually by the process of homologous recombination. Typically, the integration vector will be one which can be transferred into the target cell, but which has a replicon which is nonfunctional in that organism. Integration of the segment comprising the gene of interest may be selected if an appropriate marker is included within that segment. One or more nucleic acid sequences encoding appropriate signal peptides that are not naturally associated with a polypeptide to be expressed in a host cell of the invention can be incorporated into (expression) vectors. For example, a DNA sequence for a signal peptide leader can be fused in-frame to a nucleic acid of the invention so that the alcohol acyl transferase of the invention is initially translated as a fusion protein comprising the signal peptide. Depending on the nature of the signal peptide, the expressed polypeptide will be targeted differently. A secretory signal peptide that is functional in the intended host cells, for instance, enhances extracellular secretion of the expressed polypeptide. Other signal peptides direct the expressed polypeptide to certain organelles, like the chloroplasts, mitochondria and peroxisomes. The signal peptide can be cleaved from the polypeptide upon transportation to the intended organelle or from the cell. It is possible to provide a fusion of an additional peptide sequence at the amino or carboxyl terminal end of the polypeptide.

The term “expression construct” as used herein refers to polynucleotides comprising the polynucleotide of the invention and additional functional nucleic acid sequences. An expression construct according to the present invention is, preferably, a linear DNA molecule. Typically, an expression construct in accordance with the present invention may be a targeting construct, which allows for random or site- directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for either homologous or heterologous recombination as described in detail below. In both cases, the construct must be, preferably, impeccable, with structures to control gene expression, such as a promoter, a site of transcription initiation, a site of poly adenylation, and a site of transcription termination. Moreover, it will be understood that an expression construct in accordance with the present invention may also be generated by using genomic modification techniques such as genome editing using the CRISPR/Cas technology. The invention further contemplates a host cell comprising the polynucleotide or the vector or expression construct of the invention.

The host cell of the invention is capable of expressing the polypeptide of the invention comprised in the vector or expression construct of the invention. The host cell is, typically transformed or transduced with said vector or expression construct such that the polypeptide of the invention can be expressed from the vector or expression construct. The transformed vector or expression construct may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome as specified elsewhere herein in more detail. A host cell according to the invention may be produced based on standard genetic and molecular biology techniques that are generally known in the art, e.g., as described in standard textbooks, such as Sambrook, J., and Russell, D.W. "Molecular Cloning: A Laboratory Manual" 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001); and F.M. Ausubel et al, eds., "Current protocols in molecular biology", John Wiley and Sons, Inc., New York (1987), and later supplements thereto.

Preferably, said host cell is a bacterial cell, a fungal cell, an animal cell or a plant cell.

Bacterial cells may be gram-positive or gram-negative bacterial cells. Preferred bacterial cells may be selected from the genera Escherichia, Klebsiella, Helicobacter, Bacillus, Lactobacillus, Streptococcus, Amycolatopsis, Rhodobacter, Pseudomonas, Paracoccus, Lactococcus or Pantoea. More preferably, useful gram positive bacterial host cells may be Bacillus alkalophius, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus Jautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptomyces spheroides, Streptomyces thermoviolaceus, Streptomyces lividans, Streptomyces murinus, Streptoverticillum verticillium ssp. verticillium. Rhodobacter sphaeroides, Rhodomonas palustri, or Streptococcus lactis. In addition, more preferably, useful gram-negative bacterial host cells may be Escherichia coli, Pseudomonas sp., preferably, Pseudomonas purrocinia, Pseudomonas fluorescens, Rhodobacter capsulatus, Rhodobacter sphaeroides, Paracoccus carotinifaciens, Paracoccus zeaxanthinifaciens or Pantoea ananatis.

Preferred fungal host cells may be Aspergillus, Fusarium, Trichoderma, Yeast, Pichia, or Saccharomyces host cells. Yeast as used herein includes ascosporogenous yeast, basidiosporogenous yeast, and yeast belonging to the Blastomycetes.

Preferred animal host cells may comprise mammalian host cells, avian host cells, reptilian host cells or insect host cells. Preferred animal host cells are HeLa cells, HEK293T, F or E cells, U20S cells, A549 cells, HT1080 cells, CAD cells, P19 cells, NIH3T3 cells, L929 cells, N2a cells, CHO cells, MCF-7 cells, Y79 cells, SO-Rb50 cells, HepG2 cells, DUKX-X11 cells, J558L cells or BHK cells.

Preferred plant host cells comprise tobacco, rice, wheat, pea or tomato cells.

The present invention relates to a non-human transgenic multicellular organism comprising the polynucleotide, the vector or expression construct or the host cell of the invention.

The term “non-human transgenic organism” as used herein refers to an organism, which has been genetically modified in order to comprise the polynucleotide, vector or expression construct of the present invention. Said genetic modification may be the result of any kind of homologous or heterologous recombination event, mutagenesis or gene editing process. Accordingly, the transgenic non-human organism shall differ from its non-transgenic counterpart in that it comprises the non-naturally occurring (i.e. heterologous) polynucleotide, vector or expression construct in its genome. Non-human organisms envisaged as transgenic non-human organisms in accordance with the present invention are, preferably, multi-cellular organisms, such as an animal, plant, multi-cellular fungi or algae. Preferably, said non-human organism is an animal or a plant. Preferred animals are mammals, in particular, laboratory animals such as rodents, e.g., mice, rats, rabbits or the like, or farming animals such as sheep, goat, cows, horses or the like. Preferred plants are crop plants or vegetables, in particular, selected from the group consisting of tobacco, rice, wheat, pea and tomato. Methods for the production of transgenic non-human organisms are well known in the art; see, standard text books, e.g. Lee-Yoon Low et al., Transgenic Plants: Gene constructs, vector and transformation method. 2018. DOI.10.5772/intechopen.79369; Pinkert, C. A. (ed.) 1994. Transgenic animal technology: A laboratory handbook. Academic Press, Inc., San Diedo, Calif.; Monastersky G. M. and Robl, J. M. (ed.) (1995) Strategies in Transgenic Animal Science. ASM Press. Washington D.C); Sambrook, loc.cit, Ausubel, loc.cit).

The present invention further contemplates a method for the manufacture of a fusion protein according to the invention wherein said method comprises

(a) expressing the polynucleotide comprising the polynucleotide or the vector or expression construct of the invention in a host cell, and

(b) obtaining the said fusion protein from said host cell. The term “manufacture” as used herein refers to the process of recombinant production of the fusion protein of the invention in a host cell. The manufacture may also comprise further steps such as purifying the produced protein or formulating the said protein or purified protein as a pharmaceutical composition. Accordingly, the aforementioned method of the present invention may consist of the aforementioned steps or may comprise further additional steps.

Expressing the polynucleotide or the vector or expression construct of the invention in a host cell may, for example, also include the step of generating the polynucleotide or vector of the invention as well as the step of introducing said polynucleotide or vector or expression construct into the host cell.

Introducing the polynucleotide or vector or expression construct into a host cell for expression can be done by all techniques available in the art, including salt-based transfection, lipofection, electroporation, injection, viral transfection techniques and the like. The polynucleotide or vector or expression construct may be stably integrated into the genome of the host cell or may be transiently present.

Obtaining the fusion protein of the invention from the host cell can be achieved by purifying or partially purifying the said protein from the host cells or host cell culture. For protein purification, various techniques may be used including precipitation, filtration, ultra-filtration, extraction, chromatography techniques such as ion-exchange-, affinity- and/or size exclusion chromatography, HPLC or electrophoresis. The skilled person is well aware of how an antibody may be purified in order to provide it in isolated form. Preferred techniques are those described in the accompanying Examples below.

The present invention relates to a medicament comprising the fusion protein, the polynucleotide, the vector or expression construct or the host cell of the invention.

The term “medicament” as used herein refers to the fusion protein, polynucleotide, vector or expression construct of the invention said is formulated in a pharmaceutical acceptable manner for pharmaceutical uses. Such a medicament is, preferably, for topical or systemic administration. Conventionally a medicament will be administered intra-muscularly or subcutaneously. However, depending on the nature and the desired therapeutic effect and the mode of action, the medicament may be administered by other routes as well. In particular, in accordance with the present invention, aerosol formulations or sprays applying medicament in the respiratory systems such as the nasal tract or the lung are also conceivable. The medicament is, preferably, administered in conventional dosage forms prepared by combining the ingredients with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing or dissolving the ingredients as appropriate to the desired preparation. Preferably, a solution is envisaged for the medicament. It will be appreciated that the form and character of the pharmaceutical acceptable carrier is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well- known variables. A carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof. The pharmaceutical carrier employed may include a solid, a gel, or a liquid. Examples for solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are phosphate buffered saline solution, syrup, oil, water, emulsions, various types of wetting agents, are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution, and the like. Similarly, the carrier may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax. For polynucleotides or vectors, liposomal carriers or genetically engineered viruses may be considered as well. In particular, if a long-term application of the antibody is envisaged, a genetically engineered virus may be administered that produces the antibody of the invention over a long period within an organism to be treated. Said suitable carriers comprise those mentioned above and others well known in the art, see, e.g., standard text books such as Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. In addition, the medicament may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like. It is to be understood that the formulation of a medicament takes place under GMP standardized conditions or the like in order to ensure quality, pharmaceutical security, and effectiveness of the medicament.

A therapeutically effective dosage of the fusion protein, polynucleotide, vector or expression construct of the invention refers to an amount of said compounds to be used in medicament. A therapeutically effective dosage is an amount of a compound that prevents, ameliorates or treats the symptoms accompanying a disease or condition referred to in this specification. Therapeutic efficacy and toxicity of the compound can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment. The medicament referred to herein is administered at least once in order to treat or ameliorate or prevent a disease or condition recited in this specification. However, the said medicament may be administered more than one time.

Yet, the invention relates to a fusion protein, a polynucleotide, a vector or expression construct or a host cell for use in treating and/or preventing a disease or medical condition in a subject.

The term "treating" as used herein refers to any improvement, cure or amelioration of the disease or condition as referred to herein. It will be understood that treatment may not occur in 100% of the subjects to which the antibody has been administered. The term, however, requires that the treatment occurs in a statistically significant portion of subjects (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by a person skilled in the art using various well-known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney-U test etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.05, 0.01, 0.005, 0.001, or 0.0001.

The term “preventing” as used herein refers to significantly reducing the likelihood with which the disease or condition develops in a subject within a defined window (prevention window) starting from the administration of the antibody onwards. Typically, the prevention window is within 1 to 5 days, within 1 to 3 weeks, within 1 to 3 months or within 3 to 6 months or 3 to 12 months. However, it will be understood that the preventive window may, dependent on the kind of medicament, also be several years up to the entire lifetime. The prevention window depends on the amount of antibody, polynucleotide or vector, which is administered and the applied dosage regimen. Typically, suitable prevention windows can be determined by the clinician based on the amount of antibody or polynucleotide to be administered and the dosage regimen to be applied without further ado. It will be understood that prevention may not occur in 100% of the subjects to which the antibody has been administered. The term, however, requires that the prevention occurs in a statistically significant portion of subjects (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by a person skilled in the art using various well-known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney-U test etc. Details are described elsewhere herein.

The term “subject” as used herein relates to animals, preferably mammals, and, more preferably, humans. The subject according to the present invention shall be a subject suffering from or suspected to suffer from disease or condition as referred to herein. Typically, such a subject shows already symptoms associated with a disease or condition as referred to herein.

The term “disease or medical condition” refers to any pathophysiological condition within a subject that impairs normal functions and is typically manifested by distinguishing clinical signs and symptoms. Preferably, said disease or medical condition is cancer or an autoimmune disease.

The term “cancer” as used herein refers to a disease involving abnormal cell proliferation. Examples of diseases involving abnormal cell proliferation include, preferably, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, breast cancer, Castleman disease, cervical cancer, colon cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, rectum cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, basal and squamous cell cancer, melanoma, merkel cell cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, or Wilms tumor. The proliferatory disease can also be leukemia or lymphoma. The clinical signs and symptoms of said cancers are well known in the art and are described in standard textbooks of medicine such as Stedman.

The term “autoimmune disease” as used herein refers to a condition characterized by an abnormal immune response against the own body. Examples of autoimmune diseases, preferably, include Systemic lupus erythematosus (SLE), Goodpasture's syndrome, Sarcoidosis, Scleroderma, Rheumatoid arthritis, Dermatomyositis, Sjogren's Syndrome, Scleroderma, Dermatomyositis, Psoriasis, Vitiligo, Alopecia areata, Type 1 diabetes mellitus, Autoimmune pancreatitis, Hashimoto's thyroiditis, Addison's disease, Multiple sclerosis, Myasthenia gravis, Polyarteritis nodosa, Idiopathic thrombocytopenic purpura, Hemolytic anemia, Antiphospholipid antibody syndrome, Pernicious anemia, Gastrointestinal diseases, Celiac disease, Inflammatory bowel disease, Autoimmune hepatitis or Primary biliary cirrhosis. The clinical signs and symptoms of said autoimmune diseases are well known in the art and are described in standard textbooks of medicine such as Stedman's.

Yet, the present invention relates to a kit comprising the fusion protein, the polynucleotide or the vector or expression construct of the invention.

The term “kit” as used herein refers to a collection of components comprising, inter alia, the fusion protein, the polynucleotide or the vector or expression construct of the invention in one or more contained s). The kit shall in addition to the fusion protein, the polynucleotide or the vector or expression construct of the invention also comprise further reagents. These reagents may include components that may be useful for facilitating uptake and/or integration of the fusion protein, the polynucleotide or the vector or expression construct of the invention. Moreover, such reagents may encompass any buffer solutions required as diluents. The container also typically comprises instructions for using the compounds in a subject or in ex vivo applications such as cell culture. These instructions may be in the form of a manual or may be provided by a computer program code.

The present invention further contemplates a method for the manufacture of an IL- 15 protein wherein said method comprises

(a) expressing a polynucleotide comprising a polynucleotide or a vector or expression construct in a host cell, and

(b) obtaining the said IL- 15 protein from said host cell, wherein the IL- 15 protein has an amino acid sequence being at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1 and wherein said modified IL- 15 protein has

• an amino acid substitution of L to E at a position corresponding to position 45 of SEQ ID NO: 1,

• an amino acid substitution of N to E at a position corresponding to position 72 of SEQ ID NO: 1,

• an amino acid substitution of E to Q at a position corresponding to position 93 of SEQ ID NO: 1; and/or

• an amino acid substitution of F to L at a position corresponding to position 103 of SEQ ID NO: 1. It will be understood that the above-mentioned substitutions within the IL- 15 protein amino acid sequence will also facilitate the production of IL- 15 proteins and variants thereof lacking a fusion. The IL- 15 fusion protein or variants thereof referred to in this context can also be produced with significantly reduced aggregate formation and/or with increased production rates as specified elsewhere herein in more detail.

The explanations and definitions of the terms made above apply mutatis mutandis for the embodiments of the invention described herein below.

The following are particular preferred embodiments of the invention:

Embodiment 1 : A fusion protein comprising

(i) a binding protein that specifically binds to a cell-surface antigen on a target cell of interest, and

(ii) a modified IL- 15 protein wherein said modified IL- 15 protein has an amino acid sequence being at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1 and wherein said modified IL- 15 protein has an amino acid substitution of L to E at a position corresponding to position 45 of SEQ ID NO: 1, an amino acid substitution of N to E at a position corresponding to position 72 of SEQ ID NO: 1, an amino acid substitution of E to Q at a position corresponding to position 93 of SEQ ID NO: 1; and/or an amino acid substitution of F to L at a position corresponding to position 103 of SEQ ID NO: 1

Embodiment 2: The fusion protein of embodiment 1, wherein said modified IL- 15 protein of the fusion protein has a reduced affinity for the IL- 15 receptor alpha compared to a wildtype IL- 15 protein.

Embodiment 3: The fusion protein of embodiment 1 or 2, wherein said modified IL- 15 protein of the fusion protein is capable of binding to IL-2/IL-15 receptor beta/gamma with essentially the same affinity or higher affinity as wildtype IL- 15 protein. Embodiment 4: The fusion protein of any one of embodiments 1 to 3, wherein said modified IL- 15 protein has an amino acid substitution of E to K at a position corresponding to position 46 of SEQ ID NO: 1

Embodiment 5: The fusion protein of any one of embodiments 1 to 4, wherein said binding protein that specifically binds to a cell-surface antigen on a target cell of interest is an antibody or antibody fragment.

Embodiment 6: The fusion protein of embodiment 5, wherein said antibody or antibody fragment is an anti-CD19 antibody or antibody fragment.

Embodiment 7: The fusion protein of any one of embodiments 1 to 6, wherein said fusion protein is capable of eliciting enhanced NK cell proliferation and/or anti-tumor activity compared to a control fusion protein.

Embodiment 8: The fusion protein of embodiment 7, wherein said fusion protein comprises a modified IL- 15 protein wherein said modified IL- 15 protein has an amino acid sequence being at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1 and wherein said modified IL- 15 protein has an amino acid substitution of L to E at a position corresponding to position 45 of SEQ ID NO: 1, an amino acid substitution of N to E at a position corresponding to position 72 of SEQ ID NO: 1; and/or an amino acid substitution of E to Q at a position corresponding to position 93 of SEQ ID NO: 1.

Embodiment 9: The fusion protein of any one of embodiments 1 to 6, wherein said fusion protein exhibits reduced aggregation compared to a control fusion protein.

Embodiment 10: The fusion protein of embodiment 9, wherein said fusion protein comprises a modified IL- 15 protein wherein said modified IL- 15 protein has an amino acid sequence being at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1 and wherein said modified IL- 15 protein has an amino acid substitution of L to E at a position corresponding to position 45 of SEQ ID NO: 1, and/or an amino acid substitution of F to L at a position corresponding to position 103 of SEQ ID NO: 1. Embodiment 11 : The fusion protein of any one of claims 1 to 6 or 9, wherein said fusion protein exhibits increased production rates compared to a control fusion protein in host cells.

Embodiment 12: The fusion protein of embodiment 11 wherein said host cells are CHO cells.

Embodiment 13: The fusion protein of embodiment 11 or 12, wherein said fusion protein comprises a modified IL- 15 protein wherein said modified IL- 15 protein has an amino acid sequence being at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1 and wherein said modified IL- 15 protein has an amino acid substitution of L to E at a position corresponding to position 45 of SEQ ID NO: 1, an amino acid substitution of E to Q at a position corresponding to position 93 of SEQ ID NO: 1; and/or an amino acid substitution of F to L at a position corresponding to position 103 of SEQ ID NO: 1.

Embodiment 14: The fusion protein of any one of claims 7 to 13, wherein said control fusion protein comprises a binding protein as specified in any one of embodiments 1 to 6 and an unmodified IL- 15 protein.

Embodiment 15: The fusion protein of embodiment 14, wherein the said unmodified IL- 15 protein is an IL- 15 protein as specified in any one of embodiments 1 to 6 except that it lacks any of the amino substitutions L45E, N72E, E93Q and F103L.

Embodiment 16: A polynucleotide encoding the fusion protein of any one of embodiments 1 to 11.

Embodiment 17: The polynucleotide of embodiment 16, wherein said polynucleotide is DNA or RNA.

Embodiment 18: A vector or expression construct comprising the polynucleotide of embodiment 12 or 13.

Embodiment 19: A host cell comprising the polynucleotide of embodiment 16 or 17 or the vector or expression construct of embodiment 14. Embodiment 20: A non-human transgenic multicellular organism comprising the polynucleotide of embodiment 16 or 17, the vector or expression construct of embodiment 18 or the host cell of embodiment 19.

Embodiment 21 : A method for the manufacture of a fusion protein according to any one of embodiments 1 to 15 wherein said method comprises

(a) expressing the polynucleotide comprising the polynucleotide of embodiment 16 or 17 or the vector or expression construct of embodiment 18 in a host cell, and

(b) obtaining the said fusion protein from said host cell.

Embodiment 22: A medicament comprising the fusion protein of any one of embodiments 1 to 15, the polynucleotide of embodiment 16 or 17, the vector or expression construct of embodiment 18 or the host cell of embodiment 19.

Embodiment 23: A fusion protein of any one of embodiments 1 to 15, a polynucleotide of embodiment 16 or 17, a vector or expression construct of embodiment 18 or a host cell of embodiment 19 for use in treating and/or preventing a disease or medical condition in a subject.

Embodiment 24: The fusion protein, the polynucleotide, the vector or expression construct or the host cell for use according to embodiment 23, wherein said disease or medical condition is cancer or an autoimmune disease.

Embodiment 25: A kit comprising the fusion protein of any one of embodiments 1 to 15, the polynucleotide of embodiment 16 or 17, the vector or expression construct of embodiment 18 or the host cell of embodiment 19.

All references cited throughout this specification are, herewith, incorporated by references either in their entireties or with respect to their disclosure content specifically referred to herein.

FIGURES

Figure 1: Schematic representation of “classical” IL- 15 immunocytokines (IC 19), which were generated by fusing wildtype IL- 15 to the heavy chains of an Fc-optimized (SDIE modification) CD19 antibody (CD19mAb). Figure 2: PBMC were incubated with increasing concentrations of the indicated constructs for 72h followed by determination of (A) viable CD19⁺ B cells and (B) NK cell activation using flow cytometry. Exemplary results of one donor (right panels) and combined data (n=6 donors) obtained at 0.24 nM (left panels) are shown. P values were calculated using one-way ANOVA with Dunnett’s multiple comparison test.

Figure 3: Schematic representation of modified IL- 15 immunocytokines (MIC), which were generated by fusing an IL- 15 moiety with abrogated binding to IL-15Ra to the heavy chains of Fc-optimized (SDIE modification) antibodies (left). Such MIC constructs shall stimulate IL- 15RPy on NK cells in a target cell-restricted manner, this is, after binding of the antibody part to its target antigen (right). This is because the target cell takes over the role of the IL-15Ra- expressing cell, to which the modified protein can no longer bind.

Figure 4: Interaction interface between IL-15 and IL-15Ra. PDB ID 2Z3Q.

Figure 5: CD 19-positive NALM-6 cells were incubated with the indicated concentrations of immunocytokines consisting of CD19mAb fused to IL- 15 mutants carrying the indicated mutations in the IL- 15 moiety. Immunocytokine binding to IL- 15 receptors was assessed by incubating the formed complex (NALM-6-immunocytokine) with (A) His-tagged IL-15Ra or (B) heterodimer IL-15RPY fusion proteins followed by flow cytometry analysis.

Figure 6: PBMC were exposed to increasing concentrations of the indicated constructs for 72h and analyzed by flow cytometry for NK cell activation. Exemplary results of one donor (right panel) and combined data (n=6 donors) obtained at 0.24 nM (left panel) are shown. P values were calculated using one-way ANOVA with Dunnett’s multiple comparison test.

Figure 7: Undesired effects of non-binding IC- and MIC-proteins in vivo were analyzed using C57BL/6 mice (n= 4 mice per group) injected i.v. on 3 subsequent days with the indicated constructs (60nM). Six hours after the last injection, NK cell activation (expression of CD69 and CD25) and degranulation (CD 107a upregulation) were determined by flow cytometry.

Figure 8: PBMC were exposed to increasing concentrations of the indicated constructs for 72h followed by flow cytometric analysis of (A) CD19⁺ B cell depletion and (B) NK cell activation. Exemplary results of one donor (right panels) and combined data (n=6 donors) obtained at 0.24 nM (left panels) are shown. P values were calculated using one-way ANOVA with Dunnett’s multiple comparison test. Figure 9: CellTrace Violet labelled PBMC of healthy donors (n=4) were cultured with NALM- 6 cells at an E:T Ratio of 4: 1 in the presence or absence of the indicated constructs (1.2 nM). Medium, target cells and constructs were replenished on day 3 and 6 followed by flow cytometric analysis on day 7. (A) Experimental setup, (B) exemplary proliferation profiles and t-distributed stochastic neighbor embedding (tSNE) plots depicting resting and proliferating NK cells. (C) Division index of proliferating NK cells calculated using FlowJo software (mean values of independent experiments using PBMC of 4 different donors. P values were calculated using one-way ANOVA with Dunnett’s multiple comparison test.

Figure 10: Binding of the indicated MIC mutants to His-tagged IL-15RP and IL-15Ry as determined by ELISA. Binding graphs (A) and a table summarizing the corresponding ECso values (B) are shown (n=2).

Figure 11: The indicated MIC mutants were evaluated in the experimental setup described in Fig. 9A-9C. (A) Exemplary proliferation profiles (left) and tSNE plots (right) are shown. (B) Division index of proliferating NK cells and (C) killing of NALM-16 cells was determined by flow cytometry. Shown are mean values of results obtained in independent experiments using PBMC of 3 donors. P values were calculated using one-way ANOVA with Dunnett’s multiple comparison test.

Figure 12: Cell Trace Violet labeled PBMC of healthy donors (n=5) were cultured with NALM-6 cells expressing CD19 at an E:T Ratio of 4: 1. Medium, target cells and indicated constructs were replenished on day 3 and 6 and analyzed by flow cytometry on day 7. (A) target cell lysis, (B) NK cell proliferation are depicted. Exemplary results obtained with PBMC of one donor (left panels) and combined data (1.2 nM, n=5) (right panels) are shown. P values were calculated using one-way ANOVA with Dunnett’s multiple comparison test.

Figure 13: Cell Trace Violet labeled PBMC of healthy donors (n=5) were cultured with NALM-6 cells expressing CD20 at an E:T Ratio of 4: 1. Medium, target cells and indicated constructs were replenished on day 3 and 6 and analyzed by flow cytometry on day 7. (A) target cell lysis, (B) NK cell proliferation are depicted. Exemplary results obtained with PBMC of one donor (left panels) and combined data (1.2 nM, n=5) (right panels) are shown. P values were calculated using one-way ANOVA with Dunnett’s multiple comparison test.

Figure 14: Luciferase expressing NALM-6 cells were injected i.v. in NSG mice (n=5 per group), followed by injection of healthy human PBMC on day 3 and MIC+ 19/20 or corresponding controls (20pg each) on day 3, 5, 7 and 9. Leukemic burden was quantified by luminescence imaging at day 3 and day 10. Combined results of luminescence quantification at day 10 are shown in the right panel. Statistical analysis was performed using nonparametric Mann Whitney test.

Figure 15: (A) Primary ALL cells of two different patients and healthy PBMC as effector cells (n=3) were incubated with the indicated constructs (1.2 nM). After 72h activation of NK cells and leukemia cell lysis were determined using flow cytometry. (B) Primary ALL cells were injected i.v. into NSG mice on day 0, followed by injection of polyclonal NK cells as effectors together with MIC⁺19 and corresponding controls on day 1. Leukemic engraftment in bone marrow was analyzed by flow cytometry on day 17 and normalized to mouse CD45⁺ cells (n=5 mice per group).

Figure 16: SDS-PAGE of CD19mAb and IC19. NR: non-reduced; R: reduced.

Figure 17: The indicated constructs were subjected to analytical chromatography using Superdex S200 Increase 10/300GL column. Representative gel filtration profiles are shown.

Figure 18: Binding of the indicated constructs to CD19-expressing NALM-6 cells was determined by flow cytometry. ECso values were calculated using GraphPad Prism software.

Figure 19: SDS-PAGE of CD19mAb and the indicated IC19 mutants. NR: non-reduced; R: reduced.

Figure 20: Representative gel filtration profiles of IC19 mutants.

Figure 21: PBMC of healthy donors (n=2) were incubated with the indicated constructs with irrelevant target specificity (0.24 nM) for 72 h and cytokine release was measured by LegendPlex Assay.

Figure 22: Biochemical analysis of MIC mutants. The indicated constructs were purified by affinity chromatography and subsequently subjected to preparative size exclusion chromatography (SEC) using HiLoad 16/60 column. (A) Representative profiles are shown. (B) Table summarizing % of monomers and the corresponding yields determined by optical density and related to the volume of the supernatant used for purification.

The SEQ ID NOs referred to herein show:

SEQ ID NO: 1 : wildtype IL-15 protein, secreted; SEQ ID NO: 2: IL- 15 protein L45E, E46K;

SEQ ID NO: 3: IL- 15 protein E46K, N72E;

SEQ ID NO: 4: IL- 15 protein E46K, E93Q;

SEQ ID NO: 5: IL- 15 protein E46K, F103L;

SEQ IDNO: 6: fusion protein anti-CD19 - IL- 15 protein L45E, E46K;

SEQ ID NO: 7: fusion protein anti-CD19 - IL- 15 protein E46K, N72E;

SEQ ID NO: 8: fusion protein anti-CD19 - IL- 15 protein E46K, E93Q;

SEQ ID NO: 9: fusion protein anti-CD19 - IL- 15 protein E46K, F103L;

SEQ ID NO: 10: cDNA sequence of wildtype IL-15, secreted.

EXAMPLES

The following Examples shall merely illustrate the invention. They shall by no means construed as limiting the scope of protection sought.

Example 1: General methods and materials

Production and purification of proteins

The variable domains corresponding to CD 19 (4G7 clone), CD20 (Rituximab sequences) and isotype control (MOPC-21, GenBank no. AAD 15290.1 and AAA39002.1) as well as the human IL-15 sequence (GeneBank no: DQ893709) were codon-optimized for the transfection of CHO cells using the GeneArt GeneOptimizer tool (Thermo Fisher Scientific). VH, VL and hIL-15 sequences were synthesized de novo using GeneArt (Thermo Fisher Scientific). Construction of Fc-optimized SDIE-modified antibodies was performed as previously described (Hofmann et al, 2012). To generate immunocytokines, the human IL-15 sequence was linked to the CH3 domain via a Glycine-Serine linker (G4S)4. Mutations in IL- 15 sequences were introduced using mutagenesis PCR (Thermo Fisher Scientific). Abs were produced and purified as described previously (Zekri et al, 2021). Endotoxin levels of samples as determined by a limulus amebocyte lysate assay (Endosafe® Charles River) were < 0.5 EU/ml.

In the Figure legends and in the Examples below, abbreviations for fusion proteins are used. All fusion proteins with a designation containing “IC” comprise wildtype IL-15. All fusion proteins with a designation containing “MIC” comprise an IL- 15 variant protein which exhibits reduced binding to its IL-15Ra containing an E46K amino acid substitution. All fusion proteins with a designation containing “MIC+” contain in addition to the aforementioned E46K amino acid substitution a further amino acid substitution, L45E. Cell lines

NALM-16 and NALM-6 were purchased from DSMZ. NALM-6 cells transduced with luciferase reporter gene were generated using lentiviral vectors containing firefly luciferase under the control of EFl promoter and GFP-T2A-pac under the control of CMV promoter synthesized by VectorBuilder. Viral particles were packaged in HEK293-T cells using psPAX2 and pMD2.G packaging plasmids (Addgene). Lentiviral supernatants were collected 24-48 h after transfection and directly used for infection of NALM-6 cells. After selection with 1 pg/ml puromycin, cells were used for experiments. To validate cell authenticity, the respective immunophenotype provided by the supplier was examined on a regular basis. To exclude contamination of cultured cells with mycoplasma, cells were tested bi-weekly.

Peripheral blood samples from healthy donors and ALL patients

Peripheral blood mononuclear cells (PBMC) of healthy donors and ALL patients were obtained by density gradient centrifugation of blood samples using Ficoll/Paque (Biochrom). A written informed consent, in accordance with the Helsinki protocol, was obtained in all cases. The study was conducted according to the guidelines of the local ethics committee. Diagnosis of precursor B cell and T cell ALL was confirmed by morphologic analysis, immunophenotyping and genetic features.

ELISA and flow cytometry-based assays

Binding to IL-15RP and IL-15Ry (Biolegend) was analyzed using ELISA. Plates were coated with his-tagged IL- 15 receptors, then the indicated constructs were added at different concentrations, and binding was visualized using an HRP-conjugated goat anti-human-Fc antibody (Jackson ImmunoResearch, West Grove, PA, USA).

To define the binding affinity of the antibodies to their respective antigens, the constructs were incubated with NALM-6 cells and the binding was detected using PE-conjugated goat antihuman IgG reagents were used (Jackson ImmunoResearch). Flow cytometry was performed using the BD FACSCantoTM II and BD FACSCaliburTM systems (BD Biosciences). Data were analyzed using FlowJo (FlowJo LLC, Ashland, Oregon). EC50 values were calculated using GraphPad Prism9 (GraphPad Software). In Figure 5, CD 19-positive NALM-16 cells were incubated with the indicated concentrations of IL- 15 fusion proteins and followed by recombinant His-tagged IL-15Ra or IL-15RPy heterodimer protein (R&D Systems), followed by a biotin-tagged anti-His antibody (Qiagen) and streptavidin-PE (Life Technologies). Then cells were analyzed by flow cytometry. To analyze NK cell activation and target cell depletion, PBMCs were incubated with NALM-6 or ALL patient cells at the indicated E:T ratio in the presence or absence of the various constructs followed by flow cytometry. Activated NK cells were defined as CD3' CD56⁺CD69⁺ cells using fluorescent dye-conjugated antibodies directed to CD3, CD56 and CD69 (Biolegend). For analysis of lysis, healthy B cells and NALM-6 target cells were identified using anti-CDlO or anti-CD19 (Biolegend) antibodies, whereas primary ALL cells were identified using anti-CDlO (Biolegend). Absolute cell numbers were calculated using equal numbers of BD™CompBead (BD Biosciences). Dead cells were excluded from analysis using 7-AAD (BioLegend).

For analysis of cytokine secretion, culture supernatants were harvested after 24 h of incubation and analyzed using the CD8/NK LEGENDplex™ multiplex kits (BioLegend) according to the manufacturer's instructions.

NK cell proliferation analysis

For analysis of long-term proliferation, PBMCs were labeled with 3 pM CellTrace™ Violet dye (Thermofisher scientific) and incubated for 7 days with tumor cells and 1.2 nM of the indicated constructs. On day 3 and 6, fresh tumor cells were added to the plate together with a repetition of treatment. Cells were analyzed by flow cytometry on day 7.

Animal experiments

All animal experiments were performed following the principles of replacement, reduction and refinement (‘3R’s) according to the ARRIVE guidelines and the “European animal protection laws and policies” (Directive 2010/63/EU) with the authorization of the Institutional Animal Care and Use Committee of the University of Tubingen according to German federal and state regulations. All mouse strains were bred and maintained under specific-pathogen-free (SPF) conditions in the animal facility of the University of Tubingen.

For the in vivo toxicity model, NSG mice (NOD.Cg-Prkdcscid I12rgtmlWjl/SzI, Charles River) were injected on day 1 with human PBMC (40xl0⁶, i.p). IL-15 protein (Biolegend), ICiso or MICiso (240 nM each) were applied i.v. on day 1, 2 and 3. NK cell activation and degranulation were measured by flow cytometry in blood samples collected 6 hours after the last injection.

To evaluate efficacy, two xenograft mouse model were performed. In the first model, 25x10⁵ luciferase-transduced NALM-6 cells were injected (i.v.) on day 0. On day 3, 20xl0⁶ human PBMCs pretreated overnight with 10 ng/ml IL- 15 were injected i.v. Treatment was performed at day 3, 5, 7 and 9 (120 nM, i.v.). For monitoring leukemic burden by bioimaging, mice were injected i.p. with 100 pl D-luciferin (15 mg/ml, Biovision) and luminescence was measured using an IVIS Lumina II imager (Perkin Elmer). In the second model, 2.5xl0⁶ primary ALL patient cells were injected i.v. into mice. One day later, 5xl0⁶ polyclonal NK cells were injected i.v. together with MIC19 and the corresponding controls (140 nM). Leukemic engraftment in the bone marrow was analyzed by flow cytometry on day 17 and normalized to mouse CD45⁺ cells.

Example 2: Generation of immunocytokines with target cell-restricted IL-15 activity

IL- 15 was cloned into an expression vector previously used to produce an Fc-optimized (SDIE modification) CD 19 antibody (CD 19m Ab) (Seidel et al, 2016) to generate a “classical” immunocytokine (IC 19) (Fig. 1). After biochemical characterization (Figs. 16 and 17), flow cytometric analysis was used to ascertain that adding the IL- 15 moiety did not affect binding affinity to cell surface-expressed CD 19 (Fig. 18). CD 19 as target antigen enabled comparative evaluation of the parental CD19mAb and IC19 in an autologous setting using PBMC of healthy donors where autologous B cells served as targets for NK cell ADCC.

Compared to CD19mAb, IC19 mediated the expected significantly superior target cell lysis. An immunocytokine with irrelevant target specificity (ICiso), alike the isotype control for CD19mAb (iso), and application of recombinant IL- 15, had no effect on B cell depletion (Fig. 2A). However, no relevant difference was observed when effects of IC19 and the control construct ICiso were compared with regard to NK cell activation (Fig.2B), demonstrating that binding of the naive cytokine moiety contained in IC19 to IL-15Ra expressing cells within the PBMC induced unrestricted triggering of the IL-15Py receptor on NK cells irrespective of IC binding to its target antigen to the same extent as recombinant IL-15.

To allow for conditional IL- 15 activity, i.e. only after specific binding of the immunocytokine to target antigen, the cytokine moiety was modified in a way that binding to IL-15Ra is prevented and IL- 15 activity would be dependent on CD 19 binding (Fig. 3). The amino acids 44-50 of the IL-15-molecule are crucial for interaction with IL-15Ra (Fig. 4), and mutants with deficient IL-15Ra binding have been described (Bernard et al, 2004; Chirifu et al, 2007; Quemener et al, 2006). Taking advantage of the crystal structure of the IL-15/IL-15Ra complex (Chirifu et al, 2007), various constructs comprising mutated IL- 15 were generated (Figs. 19 and 20). Flow cytometric binding analyses revealed the desired loss of IL-15Ra binding for mutants IC19-E46K and IC19-I50D, of which IC19-E46K retained binding to IL-15RPy, and thus, was used for generation of “modified immunocytokines” (MIC) (Figs. 5A-5B).

Functional analysis using in vitro PBMC cultures revealed that MIC directed to an irrelevant target antigen (MICiso) caused significantly reduced undesired off-target NK cell activation and cytokine release compared to ICiso (Fig. 6). Taking advantage of the cross reactivity of IL- 15 in mice and humans, syngeneic C57BL/6 mice were injected with recombinant IL-15 (rlL- 15), ICiso and MICiso, and effects on NK cells by ex vivo analysis CD69 and CD25 expression as markers for activation as well as the degranulation marker CD 107a by flow cytometry were studied.

Whereas ICiso, alike rIL-15, caused significant NK cell activation and degranulation, very weak undesired effects were observed with MICiso (Fig. 7). Evaluation of MIC directed to CD 19 (MIC 19) in the aforementioned autologous setting where B cells in PBMC preparations serve as target cells revealed clearly superior target cell lysis upon treatment with MIC 19 compared to the Fc-optimized CD19mAb, while MICiso had no effect (Fig. 8 A). This was mirrored in analyses of cell activation, where MIC 19 was clearly superior to CD 19m Ab; with MICiso, NK activation was observed only at higher concentrations and to a clearly lesser extend (Fig. 8B), indicating that enhanced Fc receptor binding contributed to MIC-mediated NK cell activation.

Example 3: Optimization of the therapeutic efficacy and identification of MIC lead construct

Beyond reinforcing NK effector function, IL- 15 potently stimulates expansion and survival of NK cells, which are perquisites to combat higher tumor burden upon therapeutic intervention with NK-mobilizing treatment approaches (Waldmann, 2006). To allow for comprehensive analysis of proliferation, Celltrace-violet labeled PBMCs and NALM-6 target cells were cocultured and NK cell proliferation was determined by flow cytometry using re-challenge after 3 and 6 days to monitor long term effects (Fig. 9 A). Whereas CD 19m Ab did not induce relevant NK proliferation, this was profoundly induced by MIC 19, albeit to lesser extent than rIL-15 (Fig. 9B-C).

In principle, besides biological function of a recombinant protein its “developability”, that is producibility in an industrial production process, is essential for clinical development and, eventually, approval as a drug. To improve on the aggregation tendency and thus producibility as well as the NK cell proliferation inducing activity of MIC 19, mutants that allow for improved folding and accordingly improved producibility and superior agonism with regard to activation of IL-15PY were conceptualized. Binding analyses with eight different MIC 19 mutants (all carrying the E46K amino-acid exchange), revealed that the mutations had no undesired effect on binding affinity to IL-15RP and IL-15Ry except for MIC19-K11E showed a significant reduction of affinity (Fig. 10A-B). In addition, it is demonstrated in Fig. 22 that some of the mutations significantly reduced the aggregation tendency (measured by size exclusion chromatography) and the producibility compared to the parental MIC 19 protein, the mutant MIC19-L45E being most effective with respect to both, reduced aggregation and improved production. Moreover, in the above described long-term assay, the mutant MIC19-L45E was identified to achieve most pronounced NK cell proliferation (Figs. 11A-11B) and tumor cell killing (Fig. 11C). Based on these results, subsequent experiments were performed with MIC19 bearing the IL-15 double mutant L45E-E46K designed MIC+19.

Example 4: MIC⁺ targeting different antigens induce potent kill in vitro and in vivo

To further validate the MIC⁺ format, an additional construct directed to CD20 was generated based on Rituximab variable regions and comprising the IL- 15 double mutant L45E-E46K (MIC+20). Evaluation in the aforementioned cytometry-based long-term assays confirmed that both MIC+19 and MIC+20 by far exceeded the corresponding Fc-optimized mAbs without IL- 15 activity with regard to induction of target cell lysis and NK cell proliferation (Figs. 12 and 13).

Anti-tumor activity of both MIC⁺ was further confirmed in immunocompromised NSG mice engrafted with luciferase-expressing NALM-6 cells. Three days after engraftment, animals were adoptively transferred with human PBMC and injected with MIC+19 and MIC+20 or isotype control MIC; treatment was repeated on day 5, 7 and 9.

Tracking tumor growth by bioluminescence imaging over time revealed that both MIC⁺ molecules effectively prevented development of leukemia with statistically more pronounced effects observed for MIC+19 (Fig. 14). Efficacy of the latter was further documented using primary CD19-positive (CD20-negative) patient ALL cells as targets. MIC+19 induced pronounced and target cell restricted NK cell activation, resulting in effective killing of patient leukemia cells in vitro, which was by far more pronounced than that achieved with the clinically available Fc-optimized CD19mAb (Fig. 15A).

This was further confirmed in vivo, when patient ALL cells were injected i.v. into NSG mice, followed on the next day by injection of polyclonal NK cells as effectors and treatment with MIC+19 or control. Analysis of leukemic engraftment in bone marrow was conducted by flow cytometry on day 17 and confirmed potent and target cell-restricted activity of MIC+19 (Fig. 15B). LITERATURE

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Beck A, Wurch T, Bailly C, Corvaia N (2010). “Strategies and challenges for the next generation of therapeutic antibodies”. Nat Rev Immunol 10: 345-352 Clark RA, Kupper TS (2007). “IL- 15 and dermal fibroblasts induce proliferation of natural regulatory T cells isolated from human skin”. Blood 109: 194-202

Imamichi H, Sereti I, Lane HC (2008). “IL- 15 acts as a potent inducer of CD4(+)CD25(hi) cells expressing FOXP3”. Eur J Immunol 38: 1621-1630

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EP 3 265 478 A

WO20 13/092001

Lazar et al. (2006). “Engineered antibody Fc variants with enhanced effector function.” Proc Nat Acad Sci USA 2006; 103:4005-4010

Horton et al. (2008). "Potent in vitro and in vivo activity of an Fc-engineered anti-CD19 monoclonal antibody against lymphoma and leukemia". Cancer Res; 68:8049-8057 Foyil and Bartlett (2010) " Anti-CD30 Antibodies for Hodgkin lymphoma" Gurr Hematol Malig

Rep; 5: 140-147

W02005/085282

Claims

Claims

1. A fusion protein comprising

(i) a binding protein that specifically binds to a cell-surface antigen on a target cell of interest, and

(ii) a modified IL- 15 protein wherein said modified IL- 15 protein has an amino acid sequence being at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1 and wherein said modified IL- 15 protein has an amino acid substitution of L to E at a position corresponding to position 45 of SEQ ID NO: 1, an amino acid substitution of N to E at a position corresponding to position 72 of SEQ ID NO: 1, an amino acid substitution of E to Q at a position corresponding to position 93 of SEQ ID NO: 1; and/or an amino acid substitution of F to L at a position corresponding to position 103 of SEQ ID NO: 1

2. The fusion protein of claim 1, wherein said modified IL- 15 protein of the fusion protein has a reduced affinity for the IL- 15 receptor alpha compared to a wildtype IL- 15 protein.

3. The fusion protein of claim 1 or 2, wherein said modified IL- 15 protein of the fusion protein is capable of binding to IL-2/IL-15 receptor beta/gamma with essentially the same affinity or higher affinity as wildtype IL- 15 protein.

4. The fusion protein of any one of claims 1 to 3, wherein said modified IL-15 protein has an amino acid substitution of E to K at a position corresponding to position 46 of SEQ ID NO: 1

5. The fusion protein of any one of claims 1 to 4, wherein said binding protein that specifically binds to a cell-surface antigen on a target cell of interest is an antibody or antibody fragment, preferably, an anti-CD19 antibody or antibody fragment.

6. The fusion protein of any one of claims 1 to 5, wherein said fusion protein is capable of eliciting enhanced NK cell proliferation and/or anti-tumor activity compared to a control fusion protein.

7. The fusion protein of any one of claims 1 to 6, wherein said fusion protein exhibits reduced aggregation compared to a control fusion protein.

8. The fusion protein of any one of claims 1 to 7, wherein said fusion protein exhibits increased production rates compared to a control fusion protein in host cells, preferably, CHO cells.

9. The fusion protein of any one of claims 6 to 8, wherein said control fusion protein comprises a binding protein as specified in any one of claims 1 to 5 and an unmodified IL- 15 protein, wherein the said unmodified IL- 15 protein, preferably, is an IL- 15 protein as specified in any one of claims 1 to 6 except that it lacks any of the amino substitutions L45E, N72E, E93Q and F103L.

10. A polynucleotide encoding the fusion protein of any one of claims 1 to 9.

11. A vector or expression construct comprising the polynucleotide of claim 10.

12. A host cell comprising the polynucleotide of claim 10 or the vector or expression construct of claim 11.

13. A non-human transgenic multicellular organism comprising the polynucleotide of claim 10, the vector or expression construct of claim 11 or the host cell of claim 12.

14. A method for the manufacture of a fusion protein according to any one of claims 1 to 9 wherein said method comprises

(a) expressing the polynucleotide comprising the polynucleotide of claim 10 or the vector or expression construct of claim 11 in a host cell, and

(b) obtaining the fusion protein from said host cell.

15. A medicament comprising the fusion protein of any one of claims 1 to 9, the polynucleotide of claim 10, the vector or expression construct of claim 11 or the host cell of claim 12.

16. A fusion protein of any one of claims 1 to 9, a polynucleotide of claim 10, a vector or expression construct of claim 11 or a host cell of claim 12 for use in treating and/or preventing a disease or medical condition in a subject, wherein said disease or medical condition, preferably, is cancer or an autoimmune disease.