WO2025262329A1

WO2025262329A1 - Synthetic molecules comprising tlr2 ligands

Info

Publication number: WO2025262329A1
Application number: PCT/EP2025/067564
Authority: WO
Inventors: Christian Friedrich Wilhelm BECKER; Claudia ALTDORF; Andre Koltermann
Original assignee: SYNTAB THERAPEUTICS GmbH
Current assignee: SYNTAB THERAPEUTICS GmbH
Priority date: 2024-06-21
Filing date: 2025-06-23
Publication date: 2025-12-26
Anticipated expiration: 2026-12-21

Abstract

The present invention relates to a molecular construct comprising at least two functional entities a) and b), wherein a) is an N-formyl methionine peptide, and b) is a TLR2-ligand, wherein a) and b) are associated to one another.

Description

Synthetic molecules comprising TLR2 ligands

Reference to sequence listing submitted as a compliant xml 1.0 format file (.xml)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (see MPEP § 2442.03(a)), Rule 30 EPC, and § 11 PatV, an electronic sequence listing compliant with WIPO standard ST.26 in the form of an XML 1.0 format file is submitted concurrently with the instant application, and the entire contents of the sequence listing are incorporated herein by reference. For the avoidance of doubt, if discrepancies exist between the sequences mentioned in the specification and the electronic sequence listing, the sequences in the specification shall be deemed to be the correct ones.

FIELD OF THE INVENTION

The present application relates to molecular constructs which are useful, among others, for the treatment of diseases.

BACKGROUND

In the international application W0200800517 synthetic compounds are disclosed that comprise one or more effector moieties and one or more binder moieties, linked to one another. The effector moieties are ligands to at least one pathogen pattern recognition receptor (PRR) and the binder moieties bind to a marker of a tumor cell. Preferably, the effector moieties are ligands to a formyl peptide receptor, preferably N-formyl methionine peptides, like fMLF (N-formyl Met-Leu-Phe) or fMMYALF (N-formyl- Met-Met-Tyr-Ala-Leu-Phe). The international application W0201089019 discloses further N-formyl methionine peptides that can be used in this concept, namely fML (N-formyl-Met-Leu); fMLP (N-formyl-Met- Leu-Pro), fMLKK (N-formyl- Met-Leu-Lys-Lys); fMLPKK (N-formyl-Met-Leu-Pro-Lys- Lys) and fMLFKK (N- formyl-Met-Leu-Phe-Lys-Lys).

These peptides bind, in vivo, to the formyl peptide receptor (FPR), and serve as potent activator of the innate immune system and as immunological homing signals for sites of bacterial infection, signaling several phases of neutrophil response and activation, that can including chemoattraction, stimulation of production and release of immuno signaling molecules (e.g., interleukins, cytokines, etc.), as well as degranulation, a cellular process that includes the production and release of both chemical (e.g., hydrogen peroxide and other reactive oxygen radical species) and enzymatic agents (e.g., elastase and other digestive enzymes) capable of mediating destruction of the foreign agent or pathogen. Furthermore, these signals can activate further immune cells to fight against such bacterial infections.

The cellular response mediated by the formyl peptide receptor can include cellular polarization and transmigration, generation of superoxide O2 radicals through respiratory burst oxidase, degranulation and release of a variety of various d egrad ative enzymes, as well as phagocytosis.

These molecules have thus been suggested to act as effectors in a synthetic compound concept, where they are linked to binding moieties that are specific for a given target structure. The constructs thus achieved detect target structures, e.g., malignancy markers on a tumor cell, and activate the immune system in a target -specific manner in order to attack, or inactivate, the target structures, or the cells bearing them.

The term "effector" or “effector moiety” preferably refers to any molecular structure, which induces, controls or otherwise is part of an immunological response, of either the acquired/adaptive or the innate immune system. Effector molecules are capable of binding to a receptor, though not necessarily at the binding site of the natural ligand. Effectors can modulate signal transduction when used alone, i. e., can be surrogate ligands, or can alter signal transduction in the presence of the natural ligand. The synthetic compounds disclosed in W0200800517 and WO201089019 can thus act according to a similar principle as the well known IgG-based antibodies, which have a binding moiety (the variable region, or the CDRs comprised therein), and an effector moiety (the Fc region).

In WO2017202933 the synthetic compounds are disclosed having the specified effector moiety comprising an isoleucine residue. This effector moiety provides enhanced efficacy over the effector moieties described above. However, animal studies showed that the tumor growth inhibition induced by the synthetic compound comprising an isoleucine residue was not adequate as no significant differences between treatment and control groups in repeated in vivo studies could be observed.

This suggests that the effector moiety comprising an isoleucine residue is not sufficient to trigger a secondary immune response next to the pure activation of the first immune response.

Another effector that has been described in the art is Pam3Cys, also referred to as PAM3, Pam3-CSK4 (Pam3CysSerLys4). Pam3Cys is a synthetic triacylated lipopeptide (LP) and a ligand of Tolldike receptor 2 (TLR2 -ligand). It is a potent activator of the pro-inflammatory transcription factor NF-KB (Brand et al. 2013, Aliprantis et al. 1999). Pam3Cys mimics the acylated amino terminus of bacterial lipopolysaccharides (LPs). Bacterial LPs are a family of pro-inflammatory cell wall components found in both Gram-positive and Gram-negative bacteria. The stimulatory activity of these LPs resides in their acylated amino terminus. These bacterial LPs are recognized by TLR2, a receptor that plays a pivotal role in detecting a diverse range of pathogen-associated molecular patterns (PAMPs) (Oliveira-Nascimento et al. 2012). At the cell surface, TLR2 forms a heterodimer with co-receptors TLR1 or TLR6, depending upon either tri- or diacylation of the ligand. Once a ligand binds to either TLR2- TLR1 or TLR2-TLR6, a MyD88-dependent activation of NF-KB and AP-1 occurs, ultimately leading to an innate immune response. Recognition of Pam3Cys, a triacylated LP, is mediated by TLR2 which cooperates with TLR1 through their cytoplasmic domain to induce the signaling cascade leading to the activation of NF-KB (Ozinsky et al. 2000).

Bayik et al. (2017) showed that PAM3 can stimulate normal human monocytes to preferentially differentiate into immunosuppressive M2 -like rather than inflammatory Ml -like macrophages. Horuluoglua et al. (2019) showed that the ability of PAM3 to bias monocyte differentiation in favor of immunosuppressive macrophages may represent a novel approach to the therapy of SLE.

Horuluoglua et al. (2020) showed that PAM3 treatment reduces the severity of DSS induced colitis suggesting that PAM3 may represent a novel approach to the treatment of human inflammatory bowel disease (IBD).

It is one object of the present invention to provide novel molecular constructs that allow an improved target specific activation of a patient’s immune system.

It is one further object of the present invention to provide novel molecular constructs which open up new possibilities of the treatment of diseases.

It is one other object of the present invention to provide novel molecular constructs which allow an improvement in the treatment of neoplastic diseases, autoimmune diseases, neuropathological diseases, metabolic diseases and/or infectious diseases.

These and other objects are solved by the features of the independent claims. The dependent claims disclose embodiments of the invention which may be preferred under particular circumstances. Likewise, the specification discloses further embodiments of the invention which may be preferred under particular circumstances.

SUMMARY OF THE INVENTION

The present invention provides, among others, new entities that activate innate immune receptors such as FPRs and TLRs.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Comparison of ISEr-Y9 and ISEr-X9 in the DHR oxidation assay on human granulocytes. The share of rhodamine positive cells is a measure for the effector effect of the respective molecule. The term “ISEr” as used herein refers to Immune System Engagers. ISErs are synthetic molecular constructs utilizing the innate immune system to fight cancer cells. The high bioavailability of small ISEr molecules (5kDa to 10 kDa) leads to optimal tissue penetration. ISErs are flexible in their molecular design for multi-valency and multi-specificity and easy to produce.

ISEr-Y9 refers to a molecular construct with has binding specificity for alpha integrin and which comprises an N-formyl methionine peptide (fMIFL) as functional entity. ISEr-Y9 is disclosed in WO2017202933A1 and serves as comparison for the molecular constructs ISEr- X9 and ISEr-X59.

ISEr-X9 and ISEr-X59 refer to molecular construct comprising two functional entities, namely one N-formyl methionine peptide (fMIFL) and one TLR2 -ligand. Such constructs are also called “bifunctional constructs” herein. In a therapeutic application, the bias of the mode of action of such bifunctional constructs is on a systemic level (not excluding simultaneous activity on a tissue-specific level though, yet probably with lower activity).

The constructs may further comprise at least one binding moiety. In such case, the constructs are called “tri-(or higher-)functional constructs”. In embodiments, the binding moiety has a targeting function, for example to shuttle the construct to a cell or tissue that expresses a target to which the binding moiety binds (e.g., an inflammatory tissue, or a tumor). In a therapeutic application, the bias of the mode of action of such tri- (or higher-) functional constructs is hence on a tissue specific level (not excluding simultaneous activity on a systemic level though, yet probably with lower activity).

The at least one binding moiety is not in all embodiments essential for the functionality of the two functional entities. As mentioned above, the at least one binding moiety can be or absent, or it can be dysfunctional (e.g., a binder peptide with a scrambled amino acid sequence relative to a functional binder peptide, like e.g. sc-ISEr-X9, in which “sc” stands for “scrambled”). Constructs with a dysfunctional binding moiety would hence also be called

“bifunctional constructs” Figure 2: Comparison of ISEr-Y9 and ISEr-X9 in the DHR oxidation assay on murine blood leukocytes. Fig. 2 A and Fig. 2 B refer to two independent experiments. The share of rhodamine positive cells is a measure for the effector effect of the respective molecule.

ISEr-Y9 showed an EC50 of 30-100 nM for human (Fig. 1) cells and 500 nM for murine cells (Fig. 2). For ISEr-X9 10-fold lower efficacies were achieved. This is unexpected because ISEr-X9 has two functional entities compared to ISEr-Y9 having only one functional entity. Maximum levels of rhodamin-positive human cells in this assay achieved were 95% and 60% for murine cells, (97% for human cells withPMA, 80% for murine cells with PMA).

Figure 3: BD OptEIA ELISA for human cytokine IL-8 after stimulation of human leukocytes from one donor with different concentrations of Pam3Cys, ISEr-X9, sc-ISEr-X9, ISEr-X59, ISEr-Y9 and sc-ISEr-Y9 (all measurements were performed n=4).

Interleukin-8 (IL-8) also referred to as CXCL8 (for CXC motif chemokine 8) is an endogenous messenger substance from the group of CXC motif chemokines. CXCL8 is produced in particular by endothelial cells, monocytes, epithelial cells and fibroblasts. As an inflammatory mediator, this cytokine is involved in the chemotactic recruitment of leukocytes, in particular neutrophil granulocytes, into the inflamed tissue. In addition, CXCL8 is an angiogenesis factor and as such is involved in the formation and regeneration of blood vessels.

Figure 4: BD OptEIA ELISA for human cytokine IL-6 after stimulation of human leukocytes from one donor with different concentrations of Pam3Cys, ISEr-X9, sc-ISEr-X9, ISEr-X59, ISEr-Y9 and sc-ISEr-Y9 (all measurements were performed n=4).

Interleukin-6 (IL-6), in the past also referred to as: Interferon-p2 (IFNB2), B-cell stimulating factor, B-cell differentiation factor, liver cell stimulating factor) belongs to the interleukins (or more comprehensively to the cytokines), which regulate the inflammatory reaction of the organism. Due to the nature of its complex regulation and functions in the orchestra of other cytokines and cells, IL-6 plays a key role in the transition from mechanisms of innate immunity to mechanisms of acquired immunity within the inflammatory process. It can be seen that there is a concentration-dependent stimulation of IL-8 and IL-6 by Pam3Cys, ISEr-X9-P, sc-ISEr-X9-P, ISEr-X59-P. In contrast, both ISEr-Y9 and sc-ISEr-Y9 triggered only little stimulation of IL-8 and IL-6, irrespective of the used concentration.

Figure 5: Tumor growth curve of orthotopically implanted 4T1 cells in female Balb/c mice: Average tumor volumes (caliper measurements) of the tumors in the ISEr-Y9 treatment group (grey square) and the sc-ISEr-Y9 control group (black triangle) over 29 days. One way Anova statistical analysis of significance (*p<0.05) was performed.

Figure 6: Quantitation of the ex vivo bioluminescence measurements of primary tumors removed after 4 weeks. 4T1-Luc bearing mice were treated 7 x with 800 nmol ISEr-Y9 (grey bar) or with 800 nmol sc-ISEr-Y9 (black bar) and relative tumor sizes were measured after removal from the mice by bioluminescence.

Figure 7: Primary tumor volumes (mm3) collected during necropsies on day 18: Female BALB/c mice were implanted with 4T1 tumor cells into the left mammary fat pad of each mouse on day 0 and treated as described in the figure legend. Data are given as means + SEM, n=3.

Figure 8: Primary tumor volumes (mm3) collected during necropsies on day 18: Female BALB/c mice were implanted with 4T1 tumor cells into the left mammary fat pad of each mouse on day 0 and treated as described in the figure legend. Data are given as means + SEM, n=3. Fig. 8 A: Comparison of the negative control group (vehicle, group 1) and group 5 (10 nmol ISEr-X9) and group 8 (10 nmol sc-ISEr-X9, i.e. ISEr-X9 having a dysfunctional binder). Fig. 8 B: Comparison of the negative control group (vehicle, group 1) and group 8 (10 nmol sc-ISEr-X9).

Figures 9: Semi-quantitative pathological analysis of necrosis, vital tumor cells (vital) and infiltrating immune cells (immune cells) of formalin -fixed paraffin embedded primary tumors collected during necropsies on day 18 after hematoxylin-eosin staining: Female BALB/c mice were implanted with 4T1 tumor cells into the left mammary fat pad of each mouse on day 0. After randomization on day 14 ISEr-X9 was administered intravenously at doses of 0.08, 0.4, 2, 10 and 50 nmol daily for 5 days (groups 2-7, respectively). Additionally, the control compound sc-ISEr-X9 (dysfunctional binder) was evaluated at a dose of 10 nmol daily for 5 days (group 8). As negative control, vehicle was given intravenously daily for 5 days (group 1). Data are given as means + SD, n=3.

Figure 10: Semi-quantitative pathological analysis of necrosis, vital tumor cells (vital) and infiltrating immune cells (immune cells) of formalin -fixed paraffin embedded primary tumors collected during necropsies on day 18 after hematoxylin-eosin staining: Female BALB/c mice were implanted with 4T1 tumor cells into the left mammary fat pad of each mouse on day 0. After randomization on day 14 the control compound sc-ISEr-X9 (dysfunctional binder) was evaluated at a dose of 10 nmol daily for 5 days (group 8). As negative control, vehicle was given intravenously daily for 5 days (group 1). Data are given as means + SD, n=3.

Figure 11 : Semi-quantitative pathological analysis of necrosis, vital tumor cells (vital) and infiltrating immune cells (immune cells) of formalin -fixed paraffin embedded primary tumors collected during necropsies on day 18 after hematoxylin-eosin staining: Female BALB/c mice were implanted with 4T1 tumor cells into the left mammary fat pad of each mouse on day 0. After randomization on day 14 ISEr-X9 or the control compound sc-ISEr-X9 (dysfunctional binder) was administered intravenously at a dose of 10 nmol daily for 5 days (group 5 and 8, respectively). As negative control, vehicle was given intravenously daily for 5 days (group 1). Data are given as means + SD, n=3.

Figure 12: Multiplex analysis of several cytokines (IL-6, MIP2/CXCL-2, IL-18, MCP-l/CCL- 2, TNF-alpha and GRO alpha/CXCL-1) in sera taken 2h afterthe first treatment. ISEr-X9 was administered intravenously at doses of 0.08, 0.4, 2, 10, 50 and 200 nmol daily for 5 days (groups 2-7, respectively). Results were compared to vehicle as negative control (group 1). Data are given as means + SEM, n=3, One-way Anova statistical analysis of significance was performed (*p<0.05, ****p<0.0001).

IL-6:

The pro-inflammatory interleukin 6 plays a key role in the transition from mechanisms of innate immunity to mechanisms of acquired immunity within the inflammatory process.

MIP-2/CXCL-2: Macrophage inflammatory protein 2, which is a counterpart of human IL-8, acts as an inflammatory mediator: chemotactic recruitment of leukocytes, in particular neutrophil granulocytes, into the inflamed tissue.

MCP-1/CCL2:

Monocyte chemoattractant protein- 1 regulates migration and infiltration of monocytes and macrophages.

GRO alpha/CXCL-1 :

Neutrophil -Activating Protein 3, which is structurally related to IL-8, induces chemotaxis, shape change, increase in intracellular free calcium levels, exocytosis and the respiratory burst in neutrophil granulocytes.

IL-18:

The pro-inflammatory cytokine interleukin 18 induces IFN gamma production in T cells and natural killer cells (NK).

TNF -alpha:

Tumour necrosis factor-a (TNF-a) is a multifunctional signalling substance of the immune system that is involved in local and systemic inflammation. TNF is mainly secreted by macrophages. Its most important function is to regulate the activity of various immune cells. TNF can stimulate cell death (apoptosis), cell proliferation, cell differentiation and the release of other cytokines.

Figure 13: Multiplex analysis of several cytokines (IL-6, MIP2/CXCL-2, IL-18, MCP-l/CCL- 2, TNF -alpha and GRO alpha/CXCL-1) in sera taken 2h after the first treatment. ISEr-X9 or the control compound sc-ISEr-X9 (dysfunctional binder) was administered intravenously at a dose of 10 nmol daily for 5 days (group 5 and 8, respectively). As negative control, vehicle was given intravenously daily for 5 days (group 1). Data are given as means + SEM, n=3, One-way Anova statistical analysis of significance was performed (*p<0.05).

Figure 14: Multiplex analysis of several cytokines (IL-6, MIP2/CXCL-2, IL-18, MCP-l/CCL- 2, TNF -alpha and GRO alpha/CXCL-1) in sera taken 2h after the first treatment. The control construct sc-ISEr-X9 (dysfunctional binder) was administered intravenously at a dose of 10 nmol daily for 5 days (group 8). As negative control, vehicle was given intravenously daily for 5 days (group 1). Data are given as means + SEM, n=3, One-way Anova statistical analysis of significance was performed (*p<0.05).

DETAILED DESCRIPTION OF EMBODIMENTS

Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the devices described or process steps of the methods described, as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include singular and/or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.

Furthermore, the content of the prior art documents referred to herein is incorporated by reference. This refers, particularly, for prior art documents that disclose standard or routine methods. In that case, the incorporation by reference has mainly the purpose to provide sufficient enabling disclosure, and avoid lengthy repetitions.

According to one aspect of the invention a molecular construct is provided, which molecular construct comprises at least two functional entities a) and b), wherein a) is an N-formyl methionine peptide, and b) is a TLR2 -ligand, wherein a) and b) are associated to one another.

Such molecular construct is also called “bifunctional construct” herein. In a therapeutic application, the bias of the mode of action of such bifunctional constructs is on a systemic level (not excluding simultaneous activity on a tissue-specific level though, yet probably with lower activity).

The inventors have surprisingly found that such bifunctional construct provides improved stimulation performance compared to a molecular construct having only one functional entity (Figs. 3 and 4). The improved effect could also be shown for molecular constructs having dysfunctional binder(s). Furthermore, both molecular constructs having two such functional entities and molecular constructs having two such functional entities and dysfunctional binder(s) showed enhanced stimulation of IL-8 and IL-6 compared to Pam3Cys (Pam3CysSK4) alone, indicating a synergistic effect of the two functional entities. This finding was also unexpected because DHR oxidation assays showed that 10 -fold lower efficacies were achieved with a molecular construct having two functional entities (Figs. 1 and 2). Furthermore, in vivo studies carried out with such bifunctional construct showed a reduction in primary tumor volume already after 5 days of treatment (Figs. 7 and 8). As can be seen a reduction in primary tumor volume could be shown to a similar degree with molecular constructs having two functional entities and molecular constructs having two functional entities and dysfunctional binder(s) (Fig. 8). In contrast, even after 29 days of treatment with a molecular construct having only one functional entity, tumor growth could not be impaired (Fig 5).

As used herein, the term “TLR” refers to Toll-like-receptor. Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRR). The Toll receptor was originally discovered in Drosophila melanogaster and the mammalian homologue was named Toll-like receptor (now TLR4). TLR1, -2, -4, -5, -6, and -10 are expressed on the cell surface, whereas TLR3, -7, -8, and -9 are situated on endosomal membranes within the cell.

TLRs recognize pathogens via PAMPs (Pathogen-Associated Molecular Patterns) and therefore play a central role in innate immunity. The common structural features of all Toll- like receptors are the N-terminal leucine-rich repeat (LRR) sequences and the Toll/IL-IR homology domain (HR). The different TLRs can each identify different PAMPs through direct interaction with the respective membrane surface of the pathogen.

As used herein, the term “TLR2” refers to Toll-like-receptor 2. At the cell surface,

TLR1 and TLR2 can heterodimerize to recognize a variety of bacterial lipid structures and cell wall components, such as triacylated lipoproteins, lipoteichoic acid, and 0 -glucans. TLR2 also heterodimerizes with TLR6 to bind diacylated lipopeptides.

As used herein, the term “TLR2 -ligand” refers to any molecule, natural or synthetic, that can trigger Toll-like receptor signaling. The terms “TLR2 -ligand” and “TLR2 -agonist” can be used interchangeably. Examples for TLR-ligand are Triacylated lipoproteins, lipoteichoic acid, peptidoglycans, Zymosan, Pam3CSK4, Pam2CSK4, Diacylated lipopeptides, HSPs (heat shock protein), HMGB 1 (high-mobility group box 1), uric acid, fibronectin, ECM protein.

As used herein, the term “N -formyl methionine peptide” (abbreviated: fMet peptide) relates to a peptide which comprises, at is N-terminus, an N-formyl methionine residue. The structure ofN-formyl methionine is shown in the following, The circle marks the formyl group.

A general structure ofN-formyl methionine peptides is shown in the following.

As used herein, the term “associated to one another” refers to the association the two functional entities into one molecular construct held together by forces weaker than chemical bonds that bind atoms in molecules, or by means of a direct conjugation, e.g., by means of a covalent bond. A suitable cross-linking agent can be used therefore, e.g., a carbodiimide, like EDC or DCC. Other means of association can encompass peptide linkers, as disclosed elsewhere herein.

According to one embodiment of the invention, a molecular construct is provided, wherein the TLR2 -ligand is an acylated lipopeptide.

As used herein, the term “lipopeptide” refers to peptides to which a lipid is covalently bound. Lipopeptides (LP) can be formed by palmitoylation, myristoylation, famesylation or geranylgeranylation, among others. These modifications make them amphiphilic, i.e. they are soluble in both polar and non-polar media. Lipopeptides form micelles in aqueous solutions. Bacterial LPs are a family of pro-inflammatory cell wall components found in both Grampositive and Gram-negative bacteria.

As used herein, the term “acylated lipopeptide” refer to lipopeptides that have an acylated amino terminus. The stimulatory activity of these bacterial LPs resides in their acylated amino terminus. Bacterial LPs are recognized by TLR2.

According to one embodiment of the invention, a molecular construct is provided, wherein the TLR2 -ligand is Pam3Cys or Pam2Cys.

Pam3Cys, as used herein refers to a synthetic triacylated lipopeptide (LP), which can serve as a ligand of TLR2. The structure of Pam3Cys (A-a-Palmitoyl-k-[2,3-bis-(palmitoyloxy)-(2/?k)- propyl]-L-cystein, Palmitoyl-Cys-[(RS)-2,3-di-(palmitoyloxy)-propyl]-OH) is shown the following. The central cysteine residue is marked by the circle.

Pam2Cys, as used herein refers to synthetic triacylated lipopeptide (LP), which can serve as a ligand of TLR2. The structure of Pam2Cys (S-(2,3-bis(palmitoyloxy)propyl)-L-cysteine) is shown in the following.

It is important to note that the terms “Pam3Cys” and “Pam2Cys” are often used in the literature interchangeably with “Pam3CSK4” and “Pam2CSK4”, respectively.

Therein, SK4 designates a pentapeptide that is fused, via the N-terminal Serin residue, to the central Cy stein residue of Pam3Cys or Pam2Cys by means of a peptide bond. As discussed elsewhere, a SSGG tetrapeptide can likewise be fused to the central Cy stein residue of Pam3Cys or Pam2Cys by means of a peptide bond. Such constructs look as follows:

In contrast, when used in the molecular constructs disclosed herein, the terms “Pam3Cys” and “Pam2Cys” only refer to the respective molecules without the pentapeptide or tetrapeptide, respectively.

According to one embodiment of the invention, a molecular construct is provided, wherein the N-formyl methionine peptide has the general formula, in N->C orientation, fM(Xn), wherein fM is N-formyl methionine, while X can be any amino acid residue and n is optionally an integer between > 3 and < 12.

It needs to be noted that different X can be different amino acid residues. The different amino acid residue can be proteinogenic or non-proteinogenic.

In a preferred embodiment, the N-formyl methionine peptide is < 12 amino acids in length, more preferably < 10 amino acids in length, even more preferably < 8 amino acids in length.

In another preferred embodiment, the N-formyl methionine peptide is > 2 amino acids in length, more preferably > 3 amino acids in length, even more preferably > 4 amino acids in length.

In another preferred embodiment, the N-formyl methionine peptide of < 12 and > 2 amino acids in length, more preferably < 10 and > 3 amino acids in length, even more preferably < 8 and > 4 amino acids in length. In another preferred embodiment, the N-formyl methionine peptide comprises, in addition to the at least one isoleucine residue, at least one residue selected from the group consisting of leucine, phenylalanine, valine and/or threonine.

According to one embodiment of the invention, a molecular construct is provided, wherein the two functional entities a) and b) are associated to one another either

• directly, and/or

• via a linker.

According to one embodiment of the invention, a molecular construct is provided, wherein the linker is a linker that confers confer water solubility to the molecular construct.

Such linker is for example an oligopeptide comprising a substantial amount of amino acid residues with electrically charged side chains (Arg, His, Lys, Asp, Glu) or polar uncharged side chains (Ser, Thr, Asp, Gin).

Water solubility is one important parameter to achieve with regard to the performance of therapeutic compounds. Solubility is an important factor not only to dissipate or liquefy a substance but also to attain an optimal concentration of drug in systemic circulation required for the desired therapeutic effect. Molecules with poor solubility not only cause difficulty for in vitro and in vivo assays but also add significant burdens to drug development in the form of longer time taken and increased cost to optimize the solubility.

According to one embodiment of the invention, a molecular construct is provided, wherein the linker comprises an entity selected from the group of a) a peptide chain b) a di-, tri- or multi-block-(co) polymer c) a polyethylenglycol chain d) an antibody Fc domain e) a head. According to one embodiment of the invention, a molecular construct is provided, wherein the linker is a peptide chain having a length of between > 3 and < 10 amino acids.

According to another preferred embodiment, the linker is a peptide chain having a length of between > 3 and < 8 amino acids, more preferably between > 4 and < 6 amino acids, more preferably of 5 amino acids, more preferably of 4 amino acids.

According to one embodiment of the invention, a molecular construct is provided, wherein the linker is a peptide chain selected from the group consisting of a) SSGG (SEQ ID NO: 9), and b) SKKKK (SEQ ID NO : 10).

According to one embodiment of the invention, a molecular construct is provided, wherein the N-formyl methionine peptide comprises an isoleucine residue.

According to one embodiment of the invention, a molecular construct is provided, wherein the N-formyl methionine peptide comprises at least one amino acid sequence selected from the group consisting of fMIFL (SEQ ID NO: 1), fMLFII (SEQ ID NO: 2), fMIVTLF (SEQ ID NO: 3), fMLFIIK (SEQ ID NO: 4), fMIFTLF (SEQ ID NO: 5), fMLF (SEQ ID NO: 6), fMLP (SEQ ID NO: 7), or fML (SEQ ID NO: 8).

According to one embodiment of the invention, a molecular construct is provided, which further comprises at least one binder moiety

Such constructs are called “tri-(or higher-)functional constructs”. In embodiments, the binding moiety has a targeting function, for example to shuttle the construct to a cell or tissue that expresses a target to which the binding moiety binds (e.g., an inflammatory tissue, or a tumor). In a therapeutic application, the bias of the mode of action of such tri- (or higher-) functional constructs is hence on a tissue specific level (not excluding simultaneous activity on a systemic level though, yet probably with lower activity).

According to embodiments, the binder moiety is selected from the group consisting of

• a peptide or peptidomimetic • an antibody, or a fragment or derivative thereof

• a receptor molecule, or a fragment or derivative thereof

• an antibody mimetic, or a fragment or derivative thereof, and/or

• an aptamer, or a fragment or derivative thereof.

The term "peptide", as used herein, refers to synthetic or naturally occurring short chains of amino acid monomers linked by peptide (amide) bonds. The shortest peptides are dipeptides, consisting of 2 amino acids joined by a single peptide bond, followed by tripeptides, tetrapeptides, etc. A polypeptide is a long, continuous, and unbranched peptide chain. Peptides are distinguished from proteins on the basis of size, and as an arbitrary benchmark can be understood to contain approximately 70 or less amino acids.

The term "peptidomimetic", as used herein, refers to a compound in which chemical structures (e.g., naturally occurring amino acid residues) of a peptide have been replaced with other chemical structures which mimic the conformation the replaced structures. Examples of peptidomimetics include peptidic compounds in which the peptide backbone is substituted with one or more benzodiazepine molecules (see e. g., James et al. (1993) and "retro-inverso" peptides (see U. S. Patent NO: 4,522,752 to Sisto). The term "peptidomimetic" also refers to a molecule comprising non-naturally occurring amino acids (e.g., a D-amino acid or a modified amino acid) that conformational and functionally serve as a substitute for a particular amino acid in a peptide-containing compound without adversely interfering to a significant extent with the function of the peptide.

As used herein, the term “antibody”, shall refer to a monoclonal or polyclonal antibody, or a fragment or derivative thereof. Particularly preferred, such antibody is selected from the group consisting of IgG, IgD, IgE, IgA and/or IgM, or a fragment or derivative thereof.

As used herein, the term “fragment” shall refer to fragments of such antibody retaining, in some cases, target binding capacities, e.g.

• a CDR (complementarity determining region)

• a hypervariable region,

• a variable domain (Fv)

• a single chain variable fragment (scFv) • an IgG heavy chain (consisting of VH, CHI, hinge, CH2 and CH3 regions)

• an IgG light chain (consisting of VL and CL regions), and/or

• a Fab and/or F (ab)2

• a Nanobody or a Camelbody.

As used herein, the term “receptor molecule” is used in its widest context. The receptor molecule may be any chemical entity capable of binding to a given target. Receptor molecules include enzymes, lectins, as well as soluble or membrane bound proteins or glycoproteins to a given ligand.

Examples for such membrane bound proteins are the TNFa receptor or the VEGF receptor, fragments of which are used in the so-called Fc fusion products, e.g. in ethanercept (75 kDa extracellular domain of TNF receptor II fused to human IgGl Fc) or afflibercept (extracellular domains of VEGF receptors I and II fused to human IgGl Fc).

As used herein, the term “antibody mimetic” relates to target binding proteins which are not related to immunoglobulins. Many of the above mentioned techniques, like phase display, are applicable for these molecules as well. Such antibody mimetics are for example derived from Ankyrin Repeat Proteins, C-Type Lectins, A-domain proteins of Staphylococcus aureus, Transferrins, Lipocalins, Fibronectins, Kunitz domain protease inhibitors, Ubiquitin, Cysteine knots or knottins, thioredoxin A, and so forth, and are known to the skilled person in the art from the respective literature.

The term "aptamer", as used herein, relates to nucleic acid species which are capable of binding to molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Aptamers are useful in biotechnological and therapeutic applications as they offer molecular recognition properties that rival that of the commonly used biomolecule, antibodies. In addition to their discriminate recognition, aptamers offer advantages over antibodies or other target binders as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications. Aptamers can for example be produced through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment). While the enabling examples disclosed in the following refer to a synthetic compound in which the effector moiety is connected to a peptide or peptiomimetic binder moiety, the inventive concept is also enabled in for other types of binder moieties, as e.g., disclosed above. Obrist et al. (1983), for example, discloses a concept in which the binder moiety is a full-size IgG monoclonal antibody, while the effector moiety is fMLP, i.e., an N-formyl methionine peptide devoid of an isoleucine residue. The resulting construct is capable of evoking a local enhancement of macrophages. It is thus clear that the concept according to the invention is not only enabled with peptide binders, but also with other binder types, as set forth above.

Such full size IgG antibody has a molecular weight of about 150 Kd, and an accumulated chain length (2x heavy chain + 2x light chain) of about 1330 amino acid residues. In contrast thereto, the peptide or peptiomimetic based binder moieties for which enabling examples are disclosed herein have 10 or less amino acid residues, including non -naturally occurring amino acids. They have thus less than 1 % of the size of an IgG.

According to one embodiment of the invention, a molecular construct is provided, wherein the binder moiety is associated to the N-formyl methionine peptide and/or to the TLR2 -ligand either

• directly, and/or

• via at least one linker.

Such association can for example be accomplished by means of a direct conjugation, e.g., by means of a covalent bond. A suitable cross-linking agent can be used therefore, e.g., a carbodiimide, like EDC or DCC.

A process for conjugating an N-formyl methionine peptide to a monoclonal antibody by means of a carbodiimide is for example disclosed in Obrist et al, 1988.

A linker, in contrast, consists of a molecule or an oligo- or polymer that binds, at two different sites within the linker or at its two respective ends, the effector moiety, and the binder moiety, respectively. Preferred embodiments are discussed further down in this specification.

The term "linker" preferably refers to any structures which operably links at least one effector moiety to at least one binder. If the linker binds covalently to the effector and the binder, its minimum length is one covalent bond, e.g. one peptide bond. Other linkers comprise a variety of chemical linking and crosslinking agents including, for example, homo- or heteromultifunctional, oligo- or heterofunctional crosslinking agents. Linking or crosslinking can be achieved by any of a variety of chemistries well known in the art including, for example, activated polyethylene glycols, aldehydes, isocyanates, maleimides and the like.

As used herein, “operably linked” means that under physiological conditions of pH, ionic strength and osmotic potential, the majority of the entities of the effector and the binder are associated with each other and both entities can exhibit their intended function.

Examples for a linker of the present invention comprise, small linker, statistical coupling by homooligofunctional linker, oligo-alcohols, -amines, -carboxylic acids, thiols, defined stochiometry by heterooligofunctional coupling element, polymer (hydrophilic and lipophilic polymer), statistical coupling by homomultifunctional linker, HPMA, polylysine, hydroxyethylcellulose, hydroxyethylstarch, aminodextran, copolymers, branched polymeric scaffolds, branched PEG, dendrimers, especially polylysine dendrimers, defined stochiometry by heterooligofunctional coupling, polypeptides, functionized activated polymers, polyethyleneglycol (PEG) and/or polyurethanes.

The coupling of the units to the polymeric carrier unit, e.g. PEG, is performed using reactions known to the person skilled in the art. E.g. there are number of PEG and HES attachment methods available to those skilled in the art (see for example WO 2004/100997 giving further references, Roberts et al., 2002; U.S. Pat. NO: 4,064,118; EP 1 398 322; EP 1 398 327; EP 1 398 328; WO 2004/024761). Dimerization of molecules via PEGylation, disulfide bridges or lysine side chains is described in WO 96/40772; WO 96/40749; WO 01/38342; WO 01/091780; WO 2004/101611; WO 2004/100997; WO 2004/101600; WO 2004/101606, Wrighton et al., 1997; Johnson et al., 1997). The mentioned methods combine monomeric peptides via a linker structure in order to obtain the desired dimeric or even multimeric molecules.

According to one embodiment of the invention, the linker comprises polyethylene glycol (PEG).

As set forth above, the linkers can be straight, or branched. Branched polyethylene glycol linkers (“multi-arm-PEGs”) can be prepared as grafted copolymers or by direct polymerization initiated by a oligovalent starter. Multi-arm-PEGs are commonly prepared with free hydroxyl moieties as chain terminators or subsequently modified to yield a functionalized, activated multi-arm-PEG.

According to embodiments of the invention, the linker comprises one or more polyethylene glycol molecules of < 40 monomers in length, or > 15 monomers in length, or, preferably, between < 40 and > 15 monomers in length.

Preferably, the one or two polyethylene glycol molecules are between < 35 and > 20 monomers in length, more preferably between < 24 and > 21 monomers in length.

The choice of the suitable functional group to which polyethylene glycol may bind is based on the type of available reactive group on the amino acid residue that will be coupled to the PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine.

Preferably, the polyethylene glycol linker is bound to the effector moiety by means of one or more amino acid side chain groups. Preferably, said amino acid side chain group is an amino group. Preferably said amino acid residue which has said side chain is a lysine residue. Preferably said amino acid residue which has said side chain is located C -terminal in said effector moiety. In case two or more likers are being used, two or more amino acid residues which have said side chain are located close to one another, e.g., C -terminal.

Preferably, the effector moieties are covalently attached, e.g., via the epsilon amino groups of a lysine residue, through an amide bond to a terminal carboxy group of the polyethylene glycol linker.

According to another embodiment of the molecular construct at least two functional entities and at least one binder moiety and a linker are linked to one another by means of click chemistry.

The term “click chemistry” describes a chemistry tailored to generate substances quickly and reliably by joining small units together. Click chemistry is not a single specific reaction, but is meant to mimic nature, which also generates substances by joining small modular units. As regards the variability of molecules, many naturally occurring molecules are made from repeating units (Proteins are made from repeating amino acid units, and polysaccharides are made from repeating monosaccharide units). The connections are carbon-hetero atom bonds C-X-C, rather than carbon-carbon bonds. In addition, enzymes ensure that chemical processes can overcome large enthalpy hurdles by a series of reactions each requiring only a small energy step. Mimicking nature in organic synthesis is essential in the discovery of new pharmaceuticals given the large number of possible structures.

The size of the pool of drug candidates has been calculated at 10⁶³, based on the presumption that a candidate consists of fewer than 30 non-hydrogen atoms, weighs less than 500 daltons, is made up of atoms of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, chlorine and bromine, is stable at room temperature, and does not react with oxygen and water. Click chemistry my speed up new drug discoveries by making each reaction in a multistep synthesis fast, efficient and predictable.

Several reaction types have been identified that fit into the Click chemistry concept neatly:

• [3+2] cycloadditions, such as the Huisgen 1,3-dipolar cycloaddition (also called Azide-Alkyne Huisgen Cycloaddition)

• thiol-ene click reactions

• Diels-Alder reaction and inverse electron demand Diels-Alder reaction

• [4+1] cycloadditions between isonitriles (isocyanides) and tetrazines

• nucleophilic substitution especially to small strained rings like epoxy and aziridine compounds

• carbonyl-chemistry-like formation of ureas but not reactions of the aldol type due to low thermodynamic driving force.

• addition reactions to carbon-carbon double bonds like dihydroxylation or the alkynes in the thiol -yne reaction.

While all these types of reactions qualify as preferred embodiments, the preferred reaction type is [3+2] cycloadditions, preferably, the Azide-Alkyne Huisgen Cycloaddition. This reaction type is a 1,3-dipolar cycloaddition between an azide and a terminal or internal alkyne to give a 1,2, 3 -triazole. According to one embodiment of the invention, a molecular construct is provided, which has the following structure:

This structure shows a preferred molecular construct comprising Pam3Cys. By way of analogy, such molecular structure can also, instead of Pam3Cys, comprise Pam2Cys, or any other TLR2 ligand .

Note also that the linker shown in this structure has 5 amino acid residues, yet in embodiments it can have more or less amino acid residues. In one embodiment, the linker has 4 amino acid residues.

According to one embodiment of the invention, a molecular construct is provided, wherein the binder moiety binds to a target structure that is associated with at least one disease selected from the group consisting of

• Neoplastic diseases

• Autoimmune diseases

• Neuropathological disease,

• Metabolic diseases and/or

• Infectious diseases. The term "neoplastic disease", as used herein, refers to an abnormal state or condition of cells or tissue characterized by rapidly proliferating cell growth or neoplasm.

The term “autoimmune diseases” means a disease resulting from an immune response against a self tissue or tissue component, including both self antibody responses and cell -mediated responses.

The term "neuropathological diseases" encompasses, among others, neurodegenerative diseases, neuroinflammatoiy diseases or seizure disorders.

The term "metabolic diseases" encompasses, among others, means disorders in which errors of metabolism, imbalances in metabolism, or sub- optimal metabolism occur. The metabolic diseases as used herein also contemplate a disease that can be treated through the modulation of metabolism, although the disease itself may or may not be caused by specific metabolism blockage.

The term "infectious diseases" encompasses, among others, diseases caused by pathogens, like parasites (protozoans and metazoans), bacteria, viruses, funghi and mycoplams.

According to embodiments of the invention, the binder moiety targets a3 integrin or av06 integrin, or a subdomain or epitope thereof. Expression of a3 Integrin and/or av 6 integrin is associated with different types of cancer. The respective non-antibody compounds provide thus a promising approach to treat these diseases.

Preferably, the binder moiety comprises at least one peptide sequence selected from the group consisting of cdGY(3-NO2)GHypNc (SEQ ID NO: 11, see Table 1) or RGDLATLRQL (SEQ ID NO: 12, see Table 1), wherein c = D-Cysteine; d = D-Aspartate; Y(3-NO2) = Nitrotyrosine; Hyp = 4-Hydroxiproline, and wherein the remaining capital letters symbolize L-amino acids under the established “one letter code.” SEQ ID No 11 binds to a3 integrin, while SEQ ID No 12 binds to av06 integrin.

According to one embodiment of the invention, a molecular construct for (the manufacture of a medicament for) use in the treatment of a human or animal subject being diagnosed for, suffering from or being at risk of developing a neoplastic disease, an autoimmune disease, a neuropathological disease, a metabolic disease and/or an infectious disease is provided.

This language is deemed to encompass both the swiss type claim language accepted in some countries (in this case, brackets are deemed absent) and EPC2000 language (in this case, brackets and content within the brackets is deemed absent).

Note that this embodiment reads on cases in which the molecular construct is a bifunctional construct as well as on cases in which the molecular construct is a tri -(or higher-)functional construct. The terms “bifunctional construct” and “tri-(or higher-)functional construct are explained elsewhere herein.

In a therapeutic application, the bias of the mode of action of such bifunctional constructs is on a systemic level (not excluding simultaneous activity on a tissue-specific level though, yet probably with lower activity), while the bias of the mode of action of such tri- (or higher-) functional constructs rather on a tissue specific level (not excluding simultaneous activity on a systemic level though, yet probably with lower activity).

According to one aspect of the invention, pharmaceutical composition comprising the molecular construct according to any of the aforementioned claims, and physiologically acceptable excipients is provided.

According to one aspect of the invention, a method for treating or preventing a neoplastic disease, an autoimmune disease, a neuropathological disease, a metabolic disease and/or an infectious disease in a human or animal subject is provided, which method comprises administration of molecular construct according to the above description to a patient, in one or more therapeutically sufficient doses.

EXAMPLES

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.

DHR 123 oxidation assay

Oxidative burst in neutrophils form a part of the cells host defense, and can be evoked, e.g. by the N-formyl peptide fMLP. (Kim et al. 2003 ). Oxidation assays are thus a useful tool to determine the potency of formyl peptides. The oxidative burst activity of neutrophils was analysed in a neutrophil stimulation assay, in which the intracellular conversion of Dihydroxyrhodamin 123 (DHR) to fluorescent rhodamine was quantified (DHR oxidation assay). In short, human or murine blood leukocytes (1.25*106 cells/ml) were incubated with catalase, cytochalasin B (Sigma) and DHR (Invitrogen) in Hanks’ buffered saline (HBSS, without Ca2+, Mg2+) with 10 mM Hepes pH 7.4, 2 mM EDTA + 0.1% B SA for 10 min. at 37°C followed by incubation with ISEr-Y9 or ISEr-X9 at different concentrations for 15 min. at 37°C. PMA was used as positive control. Cell suspensions was transferred to FACS tubes already containing 83.3 pl 4% formalin. Fixed cells were kept on ice until analysis. Hoechst 33258 was added to a final concentration of 0.5 pg/ml. 10 000 cells/tube were analysed using the FACS Canto II, recording FSC, SSC and FL2 (rhodamin/PE channel). For blood derived murine leukocytes a gate was set around leukocytes and used as counting gate, to prevent too many erythrocytes being counted. Leukocytes are gated in the FSC/SSC plot; live cells are gated from the FSC/Hoechst plot, and rhodamin positive leukocytes in the PE/SSC plot. Percentages of PE-positive (rhodamin -positive) cells. Percentages of rhodamine positive neutrophils is calculated and plotted against the concentrations of the agents used.

Results are shown in Figs. 1 and 2.

Stimulation assays to show activation by TLR2 ligand

For studying activation of immune cells by TLR2 ligand human peripheral blood mononuclear cells (PBMCs) were isolated from human EDTA-blood by density centrifugation. PBMCs were incubated with lSErs or Pam3Cys (Pam3CSK4, Invivogen, stock 1 mg/ml in endotoxin-free water) as reference overnight at 37°C, 5% CO2. Supernatant was collected and a series of commercially available ELIS As was used to monitor the expression of TNF-a, IL-6, IL-8, IP- 10 and IFN-y by human Leukocytes or PBMCs of different donors incubated with ISEr-X-Pam or Pam3Cys as reference as described above. The ELISA kits used were purchased from BD Biosciences and analysis was performed according to the manufacturer’s protocols.

BD OptEIA ELISA human IL-6 (555220)

BD OptEIA ELISA human TFN-a (555212)

BD OptEIA ELISA human IP-10 (550926) BD OptEIA ELISA human IL-8 (555244) BD OptEIA ELISA human IFN-y (555142)

All standards, samples and controls were analysed in duplicates. Given the stimulation of cells in duplicates ELISA measurements of samples was n=4 for each experiment (no significant or low stimulation of IP- 10 and IFN-y, therefore not shown here).

Results are shown in Figs. 3 and 4.

Efficacy study of the test compound ISEr-Y9 and control compound sc-ISEr-Y9 in the murine breast carcinoma 4T1 tumor model implanted into the mammary fat pad of female BALB/c mice Female Balb/c mice (immunocompetent) were orthotopically (in the mammary fat pads) inoculated with 4T1-Luc cells. After 7 days, tumors were about 5 mm in diameter and therapy was started. In the treatment group (12 animals) ISEr-Y9 was intravenously (i.v.) injected 7 times every second day, at a dose of 800 nmol (about 4 mg) in a 100 pl volume (in 0,9% NaCl with 10 mM Hepes, pH 7,3) each time. Thus, the mice were treated from day 7 up to day 19 after the inoculation of tumor cells. The control group received 7 x 800 nmol sc-ISEr-Y9 (with a scrambled binder peptide, displaying no tumor cell binding, and no tumor targeting, but still containing the immune effector peptide) in parallel. After the last application, the animals were kept alive for another 10 days, to be able to follow the development of metastases. Tumor sizes as well as the development of superficial metastases were measured in regular intervals using a caliper (sizes of primary tumors) and IVIS (for primary tumors and metastases). On day 29, after the final IVIS measurement the animals were sacrificed, tumors were removed and tumor sizes ex vivo were measured by bioluminescence (IVIS).

Results are shown in Figs. 5 and 6.

PK/PD study of the test compound ISEr-X9 and control compound sc-ISEr-X9 given intravenously at different doses in the murine breast carcinoma 4T1 tumor model implanted into the mammary fat pad of female BALB/c mice

The study consisted of eight experimental groups each containing three female BALB/c mice after randomization (Groups 1-8). On day 0 0.1 x 106 4T1 tumor cells in 100 pl PBS were implanted into the left mammary fat pad of each mouse. Tumor growth was monitored by caliper measurement throughout the study. On day 14 a mean tumor volume of 298.6 mm3 was reached and tumor-bearing animals were block-randomized. After randomization on day 14, treatments were initiated on the same day. ISEr-X9 was administered intravenously at doses of 0.08, 0.4, 2, 10, 50 and 200 nmol daily for 5 days (groups 2-7, respectively). Additionally, the control compound sc-ISEr-X9 (dysfunctional binder) was evaluated at a dose of 10 nmol daily for 5 days (group 8). As negative control, vehicle was given intravenously daily for 5 days (group 1). All animals of group 7 exhibited adverse effects and had to be euthanized 1 hour after therapy on Day 16. On Day 18, 2 hours after the final dose, the study was terminated, and all remaining animals were euthanized. A necropsy was performed on all animals with analysis of tumor volume and weight ex vivo. Finally, tumor tissue was preserved as formalin-fixed paraffin-embedded (FFPE) samples for further histopathology. FFPE-tumor samples were further stained with hematoxylin-eosin for discrimination of necrosis, vital tumor cells and infiltrating immune cells.

Results are shown in Figs. 7 - 11.

Multiplex analysis of several cytokines (IL-6, MIP2/CXCL-2, IL-18, MCP-l/CCL-2, TNF-alpha and GRO alpha/CXCL-1)

Expression of cytokines is analysed on MAGPIX (Luminex) machine using a 26-Plex Cytokine & Chemokine Convenience Mouse ProcartaPlex Panel 1 Kit (Thermofisher #EXPR- 260-26088-901) including the following cytokines: Eotaxin/CCL-11, GM-CSF, GRO alpha/CXCLl, IFN gamma, IL-1 beta, IL-10, IL-12p70, IL-13, IL-17A/CTLA-8, IL-18, IL-2, IL-22, IL-23, IL-27, IL-4, IL-5, IL-6, IL-9, IP-10/CXCL10, MCP-l/CCL-2) MCP-3/CCL-7, MIP-1 alpha/CCL-3, MIP-1 beta/CCL-4, MIP-2 alpha/CXCL-2, RANTES/CCL-5 and TNF alpha. A control kit (36-Plex, Thermofisher #EPX360-26092-CTR) was used to ensure reliable performance of the kit. Analysis was performed according to the manufacturer’s protocols. All standards, samples and controls were analyzed in duplicates.

Analyzed were sera of 4T1 -tumorbearing mice of the PK/PD study taken 2h and 24h after the first application as well as from final bleeding after 5 days of treatment. A broad range of cytokines was secreted with highest levels 2h after the first application of ISEr-X9 and sc- ISEr-X9 for most of the cytokines. Shown is a selection of the most potent cytokines secreted 2h after the first application.

Results are shown in Figs. 12 - 14.

Conjugation reactions/Click chemistry

Protocol for the copper-catalyzed azide-alkyne cycloaddition of an effector-peptide carrying two alkyne groups and binder peptides carrying one azide moiety.

Reaction parameters:

1 eq alkyne 2,5 eq azide

1-6 eq CuS04 (100-200 mM solution in ddEEO)

8-50 eq sodium ascorbate (500-1500 mM solution in ddEEO)

1,2-6, 5 eq TBTA (100 mM solution in anhydrous DMF)

Reaction takes place in DMF containing 15-18% ddEEO, at room temperature, under argon atmosphere, in a 2 ml Eppendorf tube stirred by a stirring bar. Concentration of the alkyne is 2-3 mM.

All solutions are prepared freshly before starting the reaction. ddEEO is degassed and then flushed with argon before preparing the CuSCh and sodium ascorbate solution.

The reaction is stirred for 10-60 minutes.

The progress of the reaction is monitored by RP-HPLC and ESI-MS.

Reverse phase purification uses a C18 column and a linear gradient from 5-45%B in 30 minutes and the corresponding products are collected by time or mass. The buffers used are A (ddH2O + 0.05 % TFA) and B (ACN + 0.05 % TFA). The purified products are lyophilized and stored at -20 °C.

REFERENCES

• Brandt K.J. et al., 2013. TLR2 Ligands Induce NF-KB activation from endosomal compartments of human monocytes PLoS One. 8(12) :e80743.

• Aliprantis A.O. et al., 1999. Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science 285(5428) :736-9.

• Oliveira-Nascimento L. et al., 2012. The Role of TLR2 in Infection and Immunity. Front Immunol. 3 :79.

• Ozinsky A. et al.., 2000. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. PNAS. 97(25) : 13766-71.

• Vowells et al. (1995)

• Mardiney et al. (1997).

• Hennessy E. J., Parker A. E., O'Neill L. A. (2010) Targeting Toll-like receptors: emerging therapeutics? Nat. Rev. Drug Discov. 9, 293-307

• Kim et al. Journal of Immunology 171 :4425-4430 (2003)

• Bayik et al. 2017, Regulation of the maturation of human monocytes into immunosuppressive macrophages. Blood Adv. 2017 Dec 4;1(26):2510-2519.

• Horuluoglua et al. (2019), PAM3 supports the generation of M2 -like macrophages from lupus patient monocytes and improves disease outcome in murine lupus, J Autoimmun. 2019 May; 99: 24-32.

• Horuluoglua et al. (2020), PAM3 protects against DSS-induced colitis by altering the M2:M1 ratio, Sci Rep 10, 6078 (2020).

SEQUENCES

The following sequences form part of the disclosure of the present application. A WIPO ST 26 compatible electronic sequence listing is provided with this application, too. For the avoidance of doubt, if discrepancies exist between the sequences in the following table and the electronic sequence listing, the sequences in this table shall be deemed to be the correct ones.

In some cases, signal peptides may be encompassed in the reproduced sequences. In such case, the sequences shall be deemed disclosed with and without signal peptides. A readily available tool to identify signal peptides in a given protein sequence is SignalP - 6.0 provided by Dansk Technical University under https://services.healthtech.dtu.dk/service.php7SignalP. The same applies to His tags or C-Myc tags, if existing.

Table 1: Sequence listing fMet = formylated Methionine; Xal = D-Cystein; Xa2 = D-Aspartat; Xa3 = Nitrotyrosine; Xa4 = 4- Hydroxiproline

Claims

What is claimed:

1. A molecular construct comprising at least two functional entities a) and b), wherein c) is an N-formyl methionine peptide, and d) is a TLR2 -ligand, wherein a) and b) are associated to one another.

2. The molecular construct according to claim 1, wherein the TLR2 -ligand is an acylated lipopeptide.

3. The molecular construct according to claim 1 or 2, wherein the TLR2 -ligand is Pam3Cys or Pam2Cys.

4. The molecular construct according to any one of the aforementioned claims, wherein the N-formyl methionine peptide has the general formula, in N->C orientation, fM(Xn), wherein fM is N-formyl methionine, while X can be any amino acid residue and n is optionally an integer between > 3 and < 12.

5. The molecular construct according to any one of the aforementioned claims, wherein the two functional entities a) and b) are associated to one another either

• directly, and/or

• via a linker.

6. The molecular construct according to any one of the aforementioned claims, wherein the linker is a linker that confers confer water solubility to the molecular construct.

7. The molecular construct according to any one of the aforementioned claims, wherein the linker comprises an entity selected from the group of f) a peptide chain g) a di-, tri- or multi-block-(co) polymer h) a polyethylenglycol chain i) an antibody Fc domain j) a head.

8. The molecular construct according to any one of the aforementioned claims, wherein the linker is a peptide chain having a length of between > 3 and < 10 amino acids.

9. The molecular construct according to any one of the aforementioned claims, wherein the linker is a peptide chain selected from the group consisting of c) SSGG (SEQ ID NO: 9), and d) SKKKK (SEQ ID NO: 10).

10. The molecular construct according to any one of the aforementioned claims, wherein the N-formyl methionine peptide comprises an isoleucine residue.

11. The molecular construct according to any one of the aforementioned claims, wherein the N-formyl methionine peptide comprises at least one amino acid sequence selected from the group consisting of fMIFL (SEQ ID NO: 1), fMLFII (SEQ ID NO: 2), fMIVTLF (SEQ ID NO: 3), fMLFIIK (SEQ ID NO: 4), fMIFTLF (SEQ ID NO: 5), fMLF (SEQ ID NO: 6), fMLP (SEQ ID NO: 7), or fML (SEQ ID NO: 8).

12. The molecular construct according to any one of the aforementioned claims, which further comprises at least one binder moiety selected from the group consisting of

• a peptide or peptidomimetic

• an antibody, or a fragment or derivative thereof

• a receptor molecule, or a fragment or derivative thereof

• an antibody mimetic, or a fragment or derivative thereof, and/or

• an aptamer, or a fragment or derivative thereof.

13. The molecular construct according to any one of the aforementioned claims, wherein the binder moiety is associated to the N-formyl methionine peptide and/or to the TLR2 -ligand either

• directly, and/or

• via at least one linker.

14. The molecular construct according to any one of the aforementioned claims, which has the following structure:

PES— W-ffc

H ¹

15. The molecular construct according to any one of the aforementioned claims, wherein the binder moiety binds to a target structure that is associated with at least one disease selected from the group consisting of

• Neoplastic diseases

• Autoimmune diseases

• Neuropathological disease,

• Metabolic diseases and/or

• Infectious diseases.

16. The molecular construct according to any one of the aforementioned claims for (the manufacture of a medicament for) use in the treatment of a human or animal subject

• being diagnosed for,

• suffering from or

• being at risk of developing a neoplastic disease, an autoimmune disease, a neuropathological disease, a metabolic disease and/or an infectious disease.

17. A pharmaceutical composition comprising the molecular construct according to any of the aforementioned claims, and physiologically acceptable excipients

18. A method for treating or preventing a neoplastic disease, an autoimmune disease, a neuropathological disease, a metabolic disease and/or an infectious disease. in a human or animal subject, which method comprises administration of molecular construct according to any one of the aforementioned claims to a patient, in one or more therapeutically sufficient doses.