WO2024165500A1

WO2024165500A1 - Vhh-based nkp46 binders

Info

Publication number: WO2024165500A1
Application number: PCT/EP2024/052788
Authority: WO
Inventors: Lukas PEKAR; Stefan ZIELONKA; Daniel KLEWINGHAUS; Paul ARRAS; Britta LIPINSKI
Original assignee: Merck Patent Gmbh
Priority date: 2023-02-06
Filing date: 2024-02-05
Publication date: 2024-08-15

Abstract

The present disclosure relates to VHH-based NKp46 binders with favorable characteristics. Moreover, the present disclosure relates to pharmaceutical compositions comprising such a compound and the use of such compounds and such pharmaceutical compositions in medical treatment methods.

Description

VHH-BASED NKP46 BINDERS

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Despite advances in clinical treatment in the past decades, cancer is still one of the leading causes of death in the developed world. In recent years, hope for considerable progress has been raised by novel approaches that harness the power of the immune system, in particular by activating immune cells in the human body and directing them against the tumor cells. One cell type that shows great potential in this regard are natural killer cells (NK cells).

NK cells play a pivotal role in early host defense against infections and tumors. NK cells are innate immune cells that were discovered in the 1970s based on their ability to exert antitumor cell cytotoxicity without prior sensitization of the host. In contrast to T-cells that recognize distinct antigens via their variable T-cell receptors, the discrimination between healthy and stressed cells and consequently the antitumor response of NK cells is based on a sophisticated interplay between a multitude of germline-encoded activating and inhibitory receptors (Gonzales-Rodriguez et al., 2019; Chiossone et al., 2018).

Natural killer cells are innate lymphocytes that recognize discontinuity and danger in multiple tissue compartments by integrating positive and negative signals. The negative signals are generally mediated by the interaction between self MHC-I on tissues and either Killer- Immunoglobulin-like Receptor (KIR) family members or Natural Killer Group 2A (NKG2A) (Carlsten et al., 2019; Vivier et al., 2008). Positive signals are transduced via the interaction of an array of NK activation receptors including the Natural Cytotoxicity Receptors (NCRs; NKp30, NKp46, NKp44), NKG2D and DNAM-1 as well as costimulatory molecules including 4-1BB and their ligands (Koch et al., 2017; Morgado et al., 2011). For the NCRs and NKG2D, many of the ligands are ’danger signals’ that are upregulated on stressed and diseased tissues including viral infected cells and tumor cells. Another mechanism by which NK cells are activated is the bridging of the low affinity activating FcyRIIIa (CD 16a) on NK cells with cells opsonized with IgG antibodies or bispecific antibodies. Unlike the NCRs and NKG2D, signaling through FcyRIIIa is often more robust in resting NK cells but is modulated by multiple variables including functionally distinct polymorphic variants of FcyRIIIa as well as competition for binding with circulating IgG. Ultimately, the balance of activation and inhibitory signal determines whether an NK cell will become activated. As such, NK cells have an endogenous capacity to differentiate between healthy and diseased tissues.

Ultimately, NK cell activation results in target cell lysis via degranulation i.e. release of cytotoxic substances such as perforin and granzymes as well as in the production of proinflammatory cytokines and chemokines.

NK cells have shown great potential for the treatment of cancer by different approaches.

Several early clinical trials employing the adoptive transfer of wild-type or genetically modified (e.g. CAR) NK cells either alone or in combination with antibodies as a therapeutic modality for cancer have shown encouraging early results for hematological malignancies (Gonzalez- Rodriguez et al., 2019; Burger et al., 2019; Rezvani et al., 2019).

Although adoptive cell therapy with ex vivo activated NK cells represents a promising approach, the logistic complexity has also driven the development of NK directed antibody-based approaches to cancer immunotherapy. In this respect antibodies have been developed that block the interaction between inhibitory receptors on NK cells, e.g. NKG2A or KIR2DL1, KIR2DL2 or KIR2DL3, and their ligands enabling immune cell activation (Andre et al., 2018; Kohrt et al., 2014; Benson et al., 2015). Furthermore, the vast majority of NK cells express the low affinity Fey receptor CD 16a. CD16a-ligation of an antibody bound to its target cell induces potent NK cell degranulation (Bryceson et al., 2005). This process, referred to as antibodydependent cellular cytotoxicity (ADCC) is considered as one important mode of action of many therapeutic antibodies (Seidel et al., 2013). The capability of an antibody to elicit ADCC however, is affected by antigen densities on target cells. Due to the low affinity interaction of antibodies with CD 16a, low antigen densities typically result in minor degrees of opsonization and consequently in limited induction of ADCC (Koch et al., 2017). Moreover, CD16a polymorphisms in humans have been described that result in different levels of ADCC, depending on the patient’s genotype. Finally, conventional therapeutic antibodies have to compete with serum immunoglobulins for CD 16a binding, resulting in confined CD 16a occupancy and restricted ADCC capacities (Ellwanger et al., 2019).

To overcome these inherent limitations of classical antibody therapies, bi- and multispecific NK cell engagers have been developed, in which one paratope binds to activating receptor CD 16a with high affinities, while the other paratope is directed against a tumor-associated antigen (Koch et al., 2017; Rothe et al., 2015). In 2019, Vivi er and co-workers described the efficient generation of trifunctional NK cell engagers (Gauthier et al., 2019). In their work, the authors employed two activating receptors of NK cells, NKp46 as well as CD16 (Fc-mediated) for effector cell engagement. In direct comparison with rituximab and Fc-engineered obinutuzumab in mouse in vivo studies, the developed NK cell engagers were more potent supporting the notion that this class of molecules might be promising therapeutic entities for tumor treatment.

Alternatively, natural or synthetic ligands of NK cell receptors can be combined with a tumortargeting moiety in a bispecific format, thus constructing an effector cell engager. Such bispecific or trifunctional entities that form a bridge between an activating receptor on NK cells and a tumor associated antigen (TAA) on the tumor cell are referred to as NK cell engagers (Koch et al., 2017). Bispecific antibodies targeting a TAA (e.g. CD20) and NKp46, NKG2D and NKp30 (Peipp et al., 2015; Kellner et al., 2016) either via an antibody moiety or a recombinant form of the ectodomain of a ligand (e.g. ULBP2) (von Strandmann et al., 2006) have demonstrated potent target dependent cytotoxicity and cytokine release in vitro.

NKp46 (NCR1, CD335) is a member of the NCR family that is displayed by both resting and activated NK cells as well as ILC1 (innate lymphoid cells 1), and a minor T cell population. Like NKp30 and FcyRIIIA, NKp46 associates with ITAM (intracellular immunoreceptor tyrosine-based activation motif)-containing FcERI y or TCR C, chains for intracellular signal transduction. Ligands for NKp46 include hemagglutinin and other viral components, the soluble complement factor P as well as heparan sulfate proteoglycan, which is expressed by different tumors. However, a specific cellular cell surface ligand for NKp46 has not been identified yet. NKp46 is an activating receptor, and the engagement of NKp46 induces NK cell cytotoxicity and cytokine release, making NKp46 an interesting trigger molecule for immunotherapeutic intervention. Recently, bispecific antibodies engaging NKp46 and a tumor antigen, i.e. either CD19 or CD20 for targeting malignant B cells, have been generated. The authors prepared NKp46-directed paratopes based on canonical VH as well as VL comprising antigen binding sites and furthermore characterized several different sophisticated NK cell engager formats. For example, bispecific antibodies were produced in a format containing a functional Fc domain to trigger FcyRIIIA in parallel to NKp46. Trifunctional NK cell engagers (NKCE) were highly effective in inducing NK cell cytotoxicity and proved efficacy in a lymphoma xenograft model in mice. See Peipp et al., 2022. Therefore, potent strategies modulating the NKp46 axis may represent promising approaches to promote antitumor NK cell responses.

Despite the great potential of NK cell engagers, in practice their use has been limited because the available activating ligands for NK cells do not have a suitable profile of biophysical and/or functional characteristics (e.g. have an insufficient affinity for effective NK cell activation, cannot be produced economically in large amounts, do not have good stability when exposed to heat and solvents, and/or are not amenable to genetic manipulations for all desired uses, such as scaffolding, labeling, and altering specific amino acids).

Accordingly, there is a need in the art for improved ways to treat cancer. Moreover, there is a need in the art for improved ways to activate NK cells. Moreover, there is a need in the art for NK cell activating compounds, in particular for NK cell activating compounds that bind to NKp46, with improved characteristics, such as improved affinity, improved specificity, improved stability, improved manufacturability, improved amenability to genetic manipulations, improved potency and/or efficacy for the killing of tumor cells, increased effects in the release of proinflammatory cytokines, improved pharmacokinetics, reduced side effects, increased therapeutic window and/or increased patient safety. Moreover, there is a need in the art for tumor-targeted bispecific antibodies (bsAbs) that trigger NKp46-mediated tumor cell eradication. Moreover, there is a need in the art to address the above-described needs by a "standardized" approach that can be widely used for activating NK cells (such as in combination with different targeting moieties) and/or that is inexpensive and allows for fast synthetic access. The present disclosure overcomes the above-described problems and addresses the abovedescribed needs.

SUMMARY OF THE INVENTION

The present disclosure addresses the needs described above in the section "Background of the Invention" by the different aspects and embodiments described below.

The present invention is, in part, based on the surprising observation that compounds comprising a VHH antibody domain or fragment thereof as described in the present disclosure exhibit a combination of various advantageous effects. For example, advantageous effects can include (but are not limited to) a high affinity for NKp46, a high k_on rate for NKp46 binding, a low k_Off rate for NKp46 binding, a high efficiency in activating NK cells, enhanced cytotoxicity, e.g. with regard to potency and/or efficacy (in particular in the context of a molecule also including a targeting moiety) and improved manufacturability. If used in combination with an Fc region capable of FcyRIIIa binding, this cytotoxicity is further enhanced. The VHH antibody domains are very suitable for the generation of NKp46-based NKCEs (e.g. NKCEs enabling targeting of both, EGFR (epidermal growth factor receptor) and NKp46).

The VHH antibody domains of the invention allow to further augment the cytotoxic potential of NKp46-based NKCEs by format and Fc engineering (improved potency and efficacy). Bivalent targeting of NKp46 by the VHH domains of the invention was found to be beneficial in enhancing potencies for both VHHs, while bivalency for the tumor-associated antigen (TAA) EGFR significantly improved maximum killing of cells expressing EGFR at their cell surface. Consequently, bivalent targeting of both, the TAA and the trigger molecule on NK cells enabled strongest augmentation of NK cell mediated lysis capacities, while unspecific lysis of targetnegative cells was negligible.

In an aspect, the present disclosure relates to a compound comprising a VHH antibody domain or a fragment thereof, wherein

(a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHHNKp46.1, VHHNKp46.2, VHHNKp46.3, VHHNKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29, VHH NKp46.31 and VHH NKp46.34 as shown in the Table of CDRs below;

(b) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with modification, wherein the modification is that the sequence of at least one of CDR1, CDR2 and CDR3 is humanized; or

(c) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with modification, wherein the modification is

- the replacement, addition or deletion of up to three amino acids in CDR1,

- the replacement, addition or deletion of up to three amino acids in CDR2 and/or

- the replacement, addition or deletion of up to three amino acids in CDR3.

In another aspect, the present disclosure relates to a compound comprising a VHH antibody domain or a fragment thereof, wherein

(A) said VHH antibody domain comprises the VHH sequence of a VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29, VHH NKp46.31 and VHH NKp46.34 as shown in the Table of VHH Sequences below;

(B) said VHH antibody domain comprises a VHH sequence as defined in (A) with modification, wherein the modification is that said sequence is humanized; (C) said VHH antibody domain comprises a VHH sequence as defined in (A) with modification, wherein the modification is the replacement, addition or deletion of up to 25 amino acids; or

(D) said VHH antibody domain comprises a VHH sequence that is at least 75% identical to a VHH sequence referred to in (A).

(A) said VHH antibody domain consists of the VHH sequence of a VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29, VHH NKp46.31 and VHH NKp46.34 as shown in the Table of VHH Sequences;

(B) said VHH antibody domain consists of a VHH sequence as defined in (A) with modification, wherein the modification is that said sequence is humanized;

(C) said VHH antibody domain consists of a VHH sequence as defined in (A) with modification, wherein the modification is the replacement, addition or deletion of up to 25 amino acids; or

(D) said VHH antibody domain consists of a VHH sequence that is at least 75% identical to a VHH sequence referred to in (A).

In another aspect, the present disclosure relates to a pharmaceutical composition comprising the compound according to the present disclosure.

In another aspect, the present disclosure relates to a compound according to the present disclosure or a pharmaceutical composition according to the present disclosure for use as a medicament or for use in the treatment of a disease as defined below.

In another aspect, the present disclosure relates to a method for treating a disease in a patient in need thereof, comprising the step of administering to said patient a therapeutically effective amount of the compound according to the present disclosure or the pharmaceutical composition according to the present disclosure.

In another aspect, the present disclosure relates to the use of the compound according to the present disclosure or of the pharmaceutical composition according to the present disclosure for the manufacture of a medicament, preferably for the manufacture of a medicament for the treatment of a disease or disorder as defined below.

BRIEF DESCRIPTION OF THE FIGURES

In the following, reference is made to the figures. All methods referred to in the figure descriptions below were carried out as described in detail in the examples.

Figure 1 summarizes the procedure used to generate NKp46-specific single-domain antibodies (sdAbs) for NK cell redirection by camelid immunization combined with yeast surface display (YSD). (A) Schematic depiction of a bispecific SEEDbody for NK redirection based on a NKp46-specific VHH molecule in combination with a humanized version of the Fab arm of Cetuximab for EGFR targeting. The scheme was generated using PyMol software version 2.3.0. (B) FACS-based selection for the isolation of NKp46-specific VHHs by utilization of a two- dimensional staining strategy for full-length VHH display and NKp46 binding at a concentration of 1 pM. Of note, plots in the upper row show representative 10⁶ cells of the sort, while plots in the lower row show representative 5xl0⁴ cells of the corresponding sorting output in order to visualize enrichment. (C) Graphical alignment of unique amino acid sequences of 40 VHH clones obtained from YSD library sorting. CDRs are indicated. Red bars represent high sequence diversity and green bars indicate high sequence conservation at a given position. Alignment obtained using MUSCLE alignment with Geneious Prime® 2021.1.1 software.

Figure 2 shows data from fluorescence-based killing assays confirming that Fc-silenced EGFR x NKp46 NKCEs trigger NK cell-mediated lysis of EGFR-overexpressing A431 cells. (A) Fluorescence based killing assays were conducted using A431 cells and freshly isolated PBMC- derived NK cells derived from healthy donors at an effector-to-target cell (E:T) ratio of 5: 1. Bispecific NKp46-specific VHH SEEDbodies harboring a humanized version of the Fab arm of Cetuximab as well as an effector-silenced Fc region were added at a concentration of 50 nM. As positive control, the monoclonal antibody Cetuximab, activating NK cells exclusively via FcyRIIIa was included. Mean values ± SEM of four independent experiments with biological duplicates are indicated. Data was normalized to the maximum concentration of Cetuximab to allow for comparison. (B) Fluorescence based killing assays of eleven selected NKCEs in a dose-dependent manner were conducted with A431 cells and freshly isolated PBMC-derived NK cells from healthy donors at E:T = 5: 1. Cetuximab and a one-armed effector competent SEEDbody lacking the NKp46 VHH domain (oa_hu225 SEEDbody eff+) as well as the corresponding effector-silenced counterpart (oa_hu225 -SEEDbody eff-) were included as controls. Mean values ± SEM of seven independent experiments with biological duplicates are indicated. Data was normalized to the maximum concentration of Cetuximab to allow for comparison.

Figure 3 shows data from biolayer interferometry experiments and flow cytometric analysis which confirms that VHH-based NKCEs target several distinct epitopes on NKp46 and trigger significant NK cell activation. (A) Biolayer interferometry (BLI) sensograms showing competitive binding experiments of a panel of eleven NKp46 SEEDbodies for recombinant human NKp46 ECD (ECD: extracellular domain). rhNKp46 ECD (rh: recombinant human) was immobilized to the sensor tip followed by a first association step using the respective SEEDbody at a concentration of 100 nM. Subsequently, a second association step was performed using another SEEDbody at 100 nM in the presence of 100 nM first analyte. In each experiment KB buffer as well as one SEEDbody in both association steps were included as controls. (B) Representation of CD69 expression for different NKCEs and control molecules within the CD56 positive NK cell population. Percentage of activation was determined by flow cytometric analysis via simultaneous NK cell staining with CD56 PE-Cy7 and CD69 PE or respective isotype controls for appropriate gating adjustment (Fig. 8). Graphs show box whiskers plots of four independent experiments measured in biological duplicates, ns, not significant; *** p < 0.001, **** p < 0.0001 vs. oa_hu225 SEEDbody eff-.

Figure 5 summarizes data showing that format and Fc engineering approaches can be used to further augment killing capacities of VHH-based NKp46 x EGFR NKCEs. (A) Fluorescence based killing assays were conducted with EGFR-overexpressing A431 cells and freshly isolated PBMC-derived NK cells from healthy donors at an E:T ratio of 5: 1 with increasing concentrations of strictly monovalent NKp46 and EGFR targeting NKp46.2 (green) and NKp46.26 (orange) VHH SEEDbodies with effector-silenced (indicated as eff-, continuous lines and filled symbols) or effector competent (indicated as eff+, dotted lines and open symbols) Fc portions. (B) Schematic depiction of engineered antibody architectures for NK redirection based on a NKp46 specific VHH molecule in combination with humanized Cetuximab Fab in an effector silenced Fc backbone. Strictly monovalent TV-terminal fusion of NKp46 VHH and EGFR Fab shown as SEEDbody format A, while TV-terminal bivalent tandem arrangement of NKp46-specific VHHs and monovalent EGFR Fab is indicated as SEEDbody format B. TV-terminal bivalent EGFR Fab fusion with C-terminal bivalent arrangement of NKp46 VHH fused onto an IgGl backbone is indicated as design C and monovalent C-terminal NKp46 VHH fusion with bivalent TV-terminal EGFR Fab orientation is indicated as design format D. Schemes were generated using PyMol software version 2.3.0. * NKp46 VHH; # EGFR Fab. (C) Fluorescence based killing assays with NKp46.2 based NKCE formats were conducted with EGFR-overexpressing A431 cells and freshly isolated PBMC-derived NK cells from healthy donors at an E:T ratio of 5: 1 with increasing concentrations of NKp46.2 design A (green continuous line and filled symbols), design B (light blue, dotted line and open symbols), design C (blue, dotted line and open symbols) as well as design D (brown, dotted line and open symbols). (D) Fluorescence based killing assays with NKp46.26 based NKCE formats were conducted with EGFR-overexpressing A431 cells and freshly isolated PBMC-derived NK cells from healthy donors at an E:T ratio of 5: 1 with increasing concentrations of NKp46.26 design A (orange continuous line and filled symbols), design B (pink, dotted line and open symbols), design C (dark red, dotted line and open symbols) as well as design D (purple, dotted line and open symbols). For all experiments, mean values ± SEM of eight independent experiments with biological duplicates are indicated. Data was normalized to the maximum concentration of Cetuximab to allow for comparison. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. respective strictly monovalent bispecific SEEDbody eff- (design A).

Figure 5 summarizes data from biolayer interferometry assays showing that NKp46 SEEDbodies demonstrate simultaneous antigen binding capacities. Sensograms show simultaneous binding experiments to recombinant human NKp46 ECD as well as recombinant human EGFR ECD. EGFR was immobilized via its polyhistidine-tag to the sensor tip followed by a first association step using the respective SEEDbody at a concentration of 100 nM. Subsequently, a second association step was performed using rhNKp46 ECD at 100 nM. In each experiment KB buffer measurements were included as controls. Of note, NKp46.37 SEEDbody eff- is a NKp46 non-binding molecule and included as negative control for comparison. Figure 6 shows data from fluorescence-microscopy based killing assays indicating that the generated bispecific NKp46 x EGFR SEEDbodies do not mediate significant lysis of EGFR- negative target cells. Fluorescence-microscopy based killing assays with EGFR-negative ExpiCHO™ target cells and freshly isolated PBMC-derived NK effector cells at an E:T ratio of 5: E Analysis of target cell killing at 50 nM SEEDbody eff- concentration via NK cell- mediated ADCC. Reference molecule Cetuximab is included. Data was normalized to maximum tumor cell lysis mediated by 30 pM staurosporine to allow for comparison of independent experiments. Graphs show normalized means ± SEM of n = 4 different healthy donors.

Figure 7 shows data from pairwise competition assays via biolayer interferometry revealing several different epitope bins for identified NKp46 specific sdAbs. (A) Competition assays in every possible combination and orientation of the NKp46 VHH SEEDbodies enabled the identification of four epitope bins. Black boxes indicate competitive binding to NKp46, white boxes show non-competitive binding and grey boxes indicate partial competition, i.e., overlapping epitopes. (B) Epitope diagram deduced from the competition study. Epitopes are illustrated as circles. Distinct bins are indicated as bin 1 and bin 2. Overlapping circles represent (partially) competing antibodies.

Figure 8 depicts data from flow cytometric analysis showing that the sequential gating strategy enables CD69 detection on CD56-positive NK cells meditated by NKp46 x EGFR SEEDbodies. Purified NK cells from PBMCs of healthy donors were incubated with EGFR-expressing A431 target cells (E:T = 5: 1) at 37°C for 24 hours in the presence of 50 nM antibody prior staining with anti-CD56 and anti-CD69 antibodies and flow cytometric analysis. Plots in the upper row exemplarily demonstrate the gating strategy for single NK cell identification using forward and side scatter. Plots in the lower row show subsequent gating for CD56-positive cells (left) and CD69-positive cells (right) based on the depicted respective isotype controls. Each plot also includes respective percentage of gated cells.

Figure 9 shows data from size exclusion chromatography unveiling favorable purities post protein A purification of different VHH-based NKCE molecules. SEC-HPLC profiles for different formats of NKp46.2 (upper panel) and NKp46.26 (lower panel)-based bispecific NKCEs as well as the percentage of the corresponding target monomer peaks are shown. Figure 10 shows differential scanning fluorimetry data to analyze thermal unfolding of NKp46 VHH-based SEEDbodies and IgGl formats. The data confirm that there is no negative influence of molecule architecture on thermal stabilities. Overlays of the melting curves for different formats of NKp46.2 (upper panel) and NKp46.26 (lower panel)-based bispecific NKCEs were recorded utilizing a temperature gradient from 20°C to 95°C at a slope of l°C/min. First derivatives of 350 nm/330 nm curves are shown.

Figure 11 summarizes data obtained by hydrophobic interaction chromatography. The data show that format influences for NKp46 VHH-based NKCE molecules. HIC profiles for different formats of NKp46.2 (upper panel) and NKp46.26 (lower panel)-based bispecific NKCEs as well as the corresponding retention times are shown.

Figure 12 depicts results obtained by fluorescence-microscopy based killing assays and shows that NKp46-targeting VHH-based NKCEs in different molecular architectures do not trigger significant cytotoxic activities of NK cells against EGFR-negative target cells. Fluorescencemicroscopy based killing assays with EGFR-negative ExpiCHO™ target cells and freshly isolated PBMC-derived NK effector cells at an E:T ratio of 5: 1. Analysis of maximum target cell killing at 50 nM antibody concentration for all scrutinized NKCE formats, based on NKp46.2 as well as NKp46.26 VHH. Reference molecule Cetuximab is included. Data was normalized to maximum tumor cell lysis mediated by 30 pM staurosporine to allow for comparison of independent experiments. Graphs show normalized means ± SEM of n = 8 different healthy donors.

Figure 13 shows data obtained by fluorescence-based killing assays indicating that effector silenced NKCEs based on either NKp46-specific or NKp30-targeting sdAbs elicit similar NK cell-mediated lysis of EGFR-overexpressing A431 cells. Fluorescence based killing assays were conducted using A431 cells and freshly isolated PBMC-derived NK cells derived from healthy donors at an effector-to-target cell (E:T) ratio of 5: 1. Strictly monovalent bispecific NKp46 or NKp30-specific VHH SEEDbodies harboring a humanized version of the Fab arm of Cetuximab as well as an effector-silenced Fc region were analyzed in a dose-dependent manner. Mean values ± SEM of seven independent experiments with biological duplicates are indicated. Data was normalized to the maximum concentration of Cetuximab to allow for comparison. Figure 14 summarizes data from in silico sequence assessment of enriched VHH domains. sdAbs were analyzed for their sequence identity compared to the most similar human germline (MOST SIMILAR GERMLINE) either based on the entire variable chain region (SEQ-ID) or the framework region only (SEQ-ID FW) as well as for their total number of specific chemical liabilities and PTMs, i.e., non-canonical cysteins (NON-CANONICAL CYS), methionine oxidations (MET OXIDATION), deamidations or isomerizations, and N-glycosylations, in structurally exposed CDR residues as derived from automatically generated homology models. As calculated physico-chemical developability descriptors (IN SILICO PHYSCHEM), structure-based pl values (pIFv 3D), the AggScores of the entire variable regions and the AggScores of CDR regions only (CDR AggScore), as well as the Positive Patch Energy of the CDRs (CDR Positive Patch Energy) are shown. The complementing color coding indicates scores within one standard deviation from a benchmark mean (dataset of 77 biotherapeutics approved for human application) as green, scores above one standard deviation as yellow (#) and scores above two standard deviations as red (*). For the AggScores, this classification was slightly adjusted based on correlation analyses to internal experimental HIC data.

SUMMARY OF SEQUENCES

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Although the present disclosure is described in detail above and below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described by the present disclosure, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

In the following, certain elements of the present disclosure will be described in more detail, including the description of specific embodiments. However, the variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements and in any manner. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application except for where this leads to logical contradictions or the context indicates otherwise.

Unless defined otherwise herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclatures and techniques referred to in the present disclosure, e.g. nomenclatures and techniques of organic chemistry, chemical synthesis, biology, medicinal and pharmaceutical chemistry, medicine, pharmacology or toxicology, are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in the references cited and discussed throughout the present disclosure unless otherwise indicated.

According to a first aspect, the present disclosure relates to a compound comprising a VHH antibody domain or a fragment thereof, wherein

- the replacement, addition or deletion of up to three amino acids in CDR1,

- the replacement, addition or deletion of up to three amino acids in CDR3;

Table of CDRs:

"Compound", as used in the present disclosure, is not particularly limited and refers to a chemical entity of any chemical class, provided that it includes the protein domain as defined above. Thus, the compound can e.g. be an organic compound or a compound composed of an organic and an inorganic part, it can be a protein composed of a single amino acid chain, a protein composed of multiple amino acid chains that are either non-covalently or covalently associated, or a non-covalent complex including an inorganic component. The compound can consist of the amino acid sequence of the VHH antibody domain or fragment thereof alone or it can in addition include further amino acid(s) that may be covalently or non-covalently attached, or it can be associated with inorganic components. Preferably, the compound is a molecule.

For example, the compound can be a bispecific molecule comprising a VHH antibody domain according to the present disclosure linked covalently to an IgGl antibody lacking one of its "arms". Or the compound may be an antibody with a VHH antibody domain according to the present disclosure and a targeting moiety prepared in the SEED format, resulting in a bispecific antibody with a structure e.g. as shown in Fig. 1 A.

As the skilled person will understand, many other formats of the compound are possible, provided that the resulting compound does not interfere with the function of the VHH antibody domain or fragment thereof, i.e. binding to NKp46 and activation of NK cells.

The compound of the present disclosure can be prepared by standard methods of genetic engineering and recombinant protein technology known to the skilled person (see e.g. Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 2014; Coligan et al., "Current Protocols in Protein Science", 1997). Exemplary methods are also described in the Examples section of the present disclosure.

In cases where the compound cannot be expressed in a single piece, individual parts can be prepared individually and later either covalently coupled, for example by a chemical reaction with appropriate reactive groups (e.g. linkage by maleimide chemistry) or by enzymatic linkage (e.g. transglutaminase-catalyzed linkage). For example, the VHH antibody domain or fragment thereof can be prepared by recombinant protein expression and subsequently linked to an antibody or antibody fragment, resulting in a bispecific antibody compound as described in the Examples section.

If the compound comprises components that are not biomolecules (such as a peptide mimetics or a small molecule), these components may be obtained e.g. by standard methods of synthetic organic chemistry.

An "antibody" is a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or antigen binding fragment thereof, which specifically binds and recognizes an analyte (antigen). Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.

In primates such as humans, a heavy and the light chain variable domain of an antibody combine to specifically bind the antigen. Generally, a naturally occurring primate (e.g., human) or murine immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (1) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Primate antibodies can be class switched.

Certain IgG antibodies from members of the camel and dromedary (Camelus bactrianus and Calelus dromaderius) family including new world members such as llama species (Lama paccos. Lamaglama mA I.ama vicugna) of mammals as found in nature lack light chains, and are thus structurally distinct from the typical four chain quaternary structure having two heavy and two light chains, for antibodies from other animals. See PCT/EP93/02214 (WO 94/04678 published 3 March 1994). Such IgG subtypes of the llamas lack the light chains and the CHI domain and are called heavy chain antibodies. These naturally occurring camelid antibodies consisting of only a heavy chain are functional and stable in the absence of light chain. The antigen-binding site of these heavy chain antibodies is formed only by a single domain, referred to as "VHH" (Kbnning et al., 2017) or, used synonymously herein, "VHH antibody domain".

Each light and heavy chain of an antibody, contains constant domains and variable domains. References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including that of an antibody fragment. References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, such as in a primate antibody. The variable domain of a heavy chain antibody is called VHH. The VHH is composed of only one polypeptide chain of 15 kDa and is considered the smallest known natural domain with full antigen-binding capacity.

Light and heavy chain variable domains contain a "framework" region interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. The CDRs are primarily responsible for antigen binding.

The CDRs are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C- terminus), and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Light chain CDRs are sometimes referred to as CDR LI, CDR L2, and CDR L3. Heavy chain CDRs are sometimes referred to as CDR Hl , CDR H2, and CDR H3. VHH monoclonal antibodies have only a heavy chain, and thus include only one CDR1, CDR2 and CDR3. Generally, the CDR3 is primarily responsible for antigen specificity.

A VHH includes in an N- to C- direction, the following structural regions: N - FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 - C, wherein FR denotes a framework region amino acid sequence and CDR denotes a complementary determining region amino acid sequence (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991).

The extent of the framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat numbering system. In antibodies (such as primate antibodies) that include a light chain, such as a primate antibody, the CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to the Kabat numbering system. The Kabat database is now maintained online. The location of camelid CDRs can also be determined (see, for example, Sircar et al., J. Immunol. 186: 6357-6367, 2011); a program to determine camelid antibody structure, the Rosetta Antibody program, is available on the internet.

A "monoclonal antibody" is an antibody produced by a single clone of B-lymphocytes or by a cell into which the heavy chain gene (and optionally a light chain gene, such as for a primate antibody) of a single antibody have been transfected. Monoclonal antibodies may be obtained using a variety of techniques known to those skilled in the art, including standard hybridoma technology (see e.g. Kohler and Milstein, Eur. J. Immunol. (1976), vol. 5, p. 511-519; Antibodies: A Laboratory Manual, 2nd edition (2014), editor Greenfield, Cold Spring Harbor Laboratory Press (USA); Immunobiology, 5th ed. (2001), editors Janeway et al., Garland Publishing (USA)) and e.g. expression from a eukaryotic host cell transfected with a DNA molecule coding for the homogeneous antibody or from a prokaryotic host cell transfected with a DNA molecule coding for the homogeneous antibody.

VHH antibody domains can be obtained by genetic engineering to yield a small protein having high affinity for a target, resulting in a low molecular weight antibody derived protein. See e.g. Sellmann et al., 2020; U.S. Patent No. 5,759,808, issued June 2, 1998; see also Dumoulin et al., (2003); Pleschberger et al., (2003); Cortez-Retamozo et al., (2002); and Lauwereys et al., (1998).

In some embodiments, the VHH molecules can be produced as recombinant monoclonal antibodies or antigen binding fragments in different expression platforms, avoiding the use of hybridomas and mice. VHH monoclonal antibodies can be humanized monoclonal antibodies. In some embodiments, monoclonal antibodies can be chimeric antibodies.

Without being bound by theory, a VHH monoclonal antibody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers.

One consequence of the small size is the ability of the VHH monoclonal antibody to bind to antigenic sites that are functionally invisible to larger antibody proteins, such that VHH monoclonal antibodies are useful as reagents to detect antigens that are otherwise cryptic using classical immunological techniques, and thus are of use as therapeutic agents. Thus yet another consequence of small size is that a camelid VHH monoclonal antibody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody.

Without being bound by theory, low molecular weight and compact size further result in camelid VHH monoclonal antibodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. Further, these molecules can be fully expressed in prokaryotic cells such as E. coli and are expressed as fusion proteins with bacteriophage and are functional.

"Humanizing" an antib ody/antibody sequence, as used herein, refers to the process of "germlining" where a non-human (such as camelid, llama or synthetic) antibody sequence is adapted to be more similar to a human antibody sequence by replacing one or more individual amino acids with the corresponding amino acids of a human antibody sequence. Typically, as human antibody sequence will be selected that is particularly close (i.e. has a high degree of sequence homology) to the non-human sequence. Such a human antibody sequence can be identified e.g. by a BLAST search. The corresponding amino acids can then be identified by a pairwise sequence alignment between the selected human antibody sequence and the non- human antibody sequence to be humanized. After humanization, the humanized antibody still binds to the same antigen as the original non-human antibody before humanization. Humanized immunoglobulins can be constructed by means of genetic engineering. A VHH antibody domain is easily humanized based on the human VH domain, which has a sequence that is highly homologous to the sequence of the VHH antibody domain.

Furthermore, a VHH sequence can be adapted to reduce sequence liabilities. This may comprise, e.g., replacing amino acids susceptible for glycosylation, deamination, oxidization or isomerization by exchanging the respective amino acid(s) in the VHH sequence with the corresponding amino acid in the closest human germline sequence or with another amino acid (e.g. alanine). Similarly, the hydrophobic patches at the cell surface can be reduced by exchanging the respective amino acid(s) with less hydrophobic amino acid(s). Again, the amino acid replacements are chosen such that after the adaption the VHH sequence still binds to the same antigen and with similar characteristics as the VHH sequence before adaption.

VHHs can be used as modular building blocks for generating multivalent and/or multispecific antibody constructs, whereby "multivalent" means that the construct encompasses more than one single domain antibody and "multispecific" means that it encompasses single domain antibodies of more than one binding specificity.

At some occasions, the present disclosure states that a certain protein/amino acid sequence A is a "fragment" of another protein/amino acid sequence B. This means that, compared to protein/amino acid B, the protein/amino acid sequence A lacks one or more amino acids at the N-terminus and/or one or more amino acids at the C-terminus. Whether a protein/amino acid sequence lacks, compared to another protein/amino acid sequence, one or more amino acids at the N-terminus and/or one or more amino acids at the C-terminus can for example readily be determined upon forming a sequence alignment e.g. with the BLAST family of programs.

As the skilled person understands, when the present disclosure refers to an "VHH antibody domain or a fragment thereof, said fragment is an antigen-binding fragment. Thus, said fragment binds to the same antigen as the "full-length" VHH antibody domain according to the present disclosure from which said fragment is derived (namely NKp46). Preferably, said fragment of said VHH antibody domain is a C-terminal fragment. This means that compared to the "complete" VHH antibody domain sequence said fragment lacks amino acids at the N- terminus.

When the present disclosure indicates that a VHH antibody domain or fragment thereof "of one VHH selected from the group consisting of VHHNKp46.1, VHHNKp46.2 and VHHNKp46.3 as shown in the Table of CDRs" (or a corresponding wording), this means that said VHH antibody domain comprises the combination of CDRs or either VHH NKp46.1 or VHH NKp46.2 or VHH NKp46.3, but not a mixture of CDRs selected from different of the listed VHHs. Thus, said VHH antibody domain or fragment thereof includes e.g. the combination of CDR1, CDR2 and CDR3 of VHH NKp46.1 (SEQ ID NO: 42, 43 and 44), the combination of CDR1, CDR2 and CDR3 of VHH NKp46.2 (SEQ ID NO: 45, 46, and 47), or the combination of CDR1, CDR2 and CDR3 of VHH NKp46.3 (SEQ ID NO: 48, 49, and 50) etc., but not the combination of CDR1 and CDR2 of VHH NKp46.1 and CDR3 of VHH NKp46.2 (SEQ ID NO: 42, 43 and 47).

When the present disclosure defines that a VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with modification, wherein the modification is e.g. that the sequence of at least one of CDR1, CDR2 and CDR3 is humanized, the skilled person is aware that this humanization exists compared to the corresponding sequence in the Table of CDRs that provides the combinations of CDR sequences without modification.

If in the definition above the present disclosure indicates the existence of a modification which is "replacement, addition or deletion" of a certain number of amino acids (e.g. up to three), the skilled person understands that this is an individual replacement, addition or deletion. Thus, the replaced, added or deleted amino acids may be at neighboring positions or at independent, isolated positions within the amino acid sequence. Moreover, as above, the skilled person is aware that this definition indicates the replacement, addition or deletion compared to the unmodified sequence in the Table of CDRs.

In some embodiments, the modification in (b) is that the sequence of CDR1 and/or CDR2, but not the sequence of CDR3 is humanized.

In some embodiments, the modification in (b) is that the sequence of CDR1 is humanized, but not the sequence of CDR2 and CDR3.

In some embodiments, the modification in (b) is that the sequence of CDR2 is humanized, but not the sequence of CDR1 and CDR3.

In some embodiments, the modification in (b) is that the sequence of one, but not more than one of CDR1, CDR2 and CDR3 is humanized.

In some embodiments, said humanization of said CDR(s) is by replacing at least one amino acid in the sequence of said CDR by the corresponding amino acid of a human VH domain.

In some embodiments, said humanization of said CDR(s) is by replacing up to three amino acids in the sequence of said CDR by the corresponding amino acid of a human VH domain.

In some embodiments, said humanization of said CDR(s) is by replacing up to three amino acids in the sequence of CDR1 and/or CDR2 and up to one amino acid in the sequence of CDR3 by the corresponding amino acid of a human VH domain.

In some embodiments, said humanization of said CDR(s) is by replacing up to two amino acids in the sequence of said CDR by the corresponding amino acid of a human VH domain.

In some embodiments, said humanization of said CDR(s) is by replacing up to two amino acids in the sequence of CDR1 and/or CDR2 and up to one amino acid in the sequence of CDR3 by the corresponding amino acid of a human VH domain. In some embodiments, said humanization of said CDR(s) is by replacing one amino acid in the sequence of said CDR by the corresponding amino acid of a human VH domain.

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to three amino acids in CDR1,

- the replacement, addition or deletion of up to one amino acid in CDR3.

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to two amino acids in CDR1,

- the replacement, addition or deletion of up to two amino acids in CDR2 and/or

- the replacement, addition or deletion of up to two amino acids in CDR3;

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to two amino acids in CDR1;

- the replacement, addition or deletion of up to two amino acids in CDR2; and/or

- the replacement, addition or deletion of up to one amino acid in CDR3.

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to two amino acids in CDR1 and/or

- the replacement, addition or deletion of up to two amino acids in CDR2; wherein the sequence of CDR3 is unmodified.

As a skilled person understands, the indication that the "sequence of CDR3 is unmodified" means that the sequence is unmodified compared to the sequence provided for CDR3 for the VHH at issue in the Table of CDRs.

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to two amino acids in CDR1, wherein the sequence of CDR2 and CDR3 is unmodified.

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to two amino acids in CDR2, wherein the sequence of CDR1 and CDR3 is unmodified. In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to one amino acid in CDR1;

- the replacement, addition or deletion of up to one amino acid in CDR2; and/or

- the replacement, addition or deletion of up to one amino acid in CDR3.

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of one amino acid in CDR1 and/or

- the replacement, addition or deletion of one amino acid in CDR2; wherein the sequence of CDR3 is unmodified.

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of one amino acid in CDR1, wherein the sequence of CDR2 and CDR3 is unmodified.

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of one amino acid in CDR2, wherein the sequence of CDR1 and CDR3 is unmodified.

In some embodiments, the modification in (c) comprises only the replacement, but not the addition or deletion of amino acids. In some embodiments, said replacement is a conservative amino acid replacement.

As used herein, a "conservative amino acid replacement" refers to the replacement of an amino acid by another, biologically similar amino acid. Conservative replacements are not likely to change the shape or characteristics of a protein/amino acid sequence. Examples of conservative replacements include the replacement of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.

According to a second aspect, the present disclosure relates to a compound comprising a VHH antibody domain or a fragment thereof, wherein

(B) said VHH antibody domain comprises a VHH sequence as defined in (A) with modification, wherein the modification is that said sequence is humanized;

(C) said VHH antibody domain comprises a VHH sequence as defined in (A) with modification, wherein the modification is the replacement, addition or deletion of up to 25 amino acids; or

(D) said VHH antibody domain comprises a VHH sequence that is at least 75% identical to a VHH sequence referred to in (A);

Table of VHH Sequences:

According to a third aspect, the present disclosure relates to a compound comprising a VHH antibody domain or a fragment thereof, wherein

(D) said VHH antibody domain consists of a VHH sequence that is at least 75% identical to a VHH sequence referred to in (A). To the second and third aspects the same explanations and definitions provided with respect to the first aspect above apply accordingly.

As the skilled person is aware, if the present disclosure indicates that a VHH antibody domain "comprises the VHH sequence of a VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH

NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH

NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH

NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH NKp46.26, VHH NKp46.27, VHH

NKp46.28, VHH NKp46.29, VHH NKp46.31 and VHH NKp46.34 as shown in the Table of VHH Sequences", this means that said VHH antibody domain comprises one and (not multiple or all) of the sequences listed in the Table of VHH Sequences.

Thus, if the present disclosure indicates that a VHH antibody domain or fragment thereof comprises/consists of a VHH sequence as defined in (A) with modification, wherein the modification is that said sequence is humanized, the skilled person is aware that this means that this humanization exists compared to the corresponding sequence in the Table of VHH Sequences that provides the sequences without modification.

If in the definition above the present disclosure indicates the existence of a modification which is "replacement, addition or deletion" of a certain number of amino acids (e.g. up to 25 amino acids), the skilled person understands that this is an individual replacement, addition or deletion. Thus, the replaced, added or deleted amino acids may be at neighboring positions or at independent, isolated positions within the amino acid sequence. Moreover, as above, the skilled person is aware that this definition indicates the replacement, addition or deletion compared to the unmodified sequence in the Table of VHH Sequences.

If the present disclosure states that a certain sequence A "is at least x % identical" to another sequence B, this is synonymous to the statement that sequence A "has x % identity" to sequence B. The statement reflects a relationship between the two polypeptide sequences A and B determined by comparing the sequences. In general, identity refers to an exact amino acid to amino acid correspondence of the two polypeptide sequences, respectively, over the length of the sequences being compared. For sequences where there is not an exact correspondence, a percentage to which the two sequences are identical may be determined. In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.

Methods for comparing the identity of two or more sequences are well known in the art. Thus, for instance, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux J et al., 1984), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity between two polypeptide sequences. BESTFIT uses the "local homology" algorithm of Smith and Waterman (1981) and finds the best single region of similarity between two sequences. Other programs for determining identity sequences are also known in the art, for instance the BLAST family of programs (Altschul S F et al, 1990, Altschul S F et al, 1997, accessible through the home page of the NCBI at www.ncbi.nlm.nih.gov) and FASTA (Pearson WR, 1990). Preferably, % identity according to the present disclosure is determined according to the BLAST family of programs (Altschul S F et al, 1990, Altschul S F et al, 1997, accessible through the home page of the NCBI at www.ncbi.nlm.nih.gov).

In some embodiments, said fragment of said VHH antibody domain comprises at least 75% of the amino acids of the sequence of said VHH antibody domain. As the skilled person understands, this means that that fragment lacks up to a quarter of the total number of amino acids of said VHH antibody domain, wherein, compared to the "complete" VHH antibody domain sequence said amino acids are lacking either at the N-terminus or at the C-terminus.

In some embodiments, said fragment of said VHH antibody domain comprises at least 80% of the amino acids of the sequence of said VHH antibody domain.

In some embodiments, said fragment of said VHH antibody domain comprises at least 85% of the amino acids of the sequence of said VHH antibody domain. In some embodiments, said fragment of said VHH antibody domain comprises at least 90% of the amino acids of the sequence of said VHH antibody domain.

In some embodiments, said fragment of said VHH antibody domain comprises at least 95% of the amino acids of the sequence of said VHH antibody domain.

In some embodiments, said fragment of said VHH antibody domain comprises at least 98% of the amino acids of the sequence of said VHH antibody domain.

In some embodiments, said fragment of said VHH antibody domain comprises at least 99% of the amino acids of the sequence of said VHH antibody domain.

In some embodiments, said fragment of said VHH antibody domain comprises complementarity determining regions CDR1, CDR2 and CDR3.

In some embodiments, said fragment of said VHH antibody domain comprises at least the sequence from the N-terminus of CDR1 to the C-terminus of CDR3 of said VHH antibody domain.

In some embodiments, in (A) said fragment of said VHH antibody domain comprises all the complementarity determining regions (CDRs) of said VHH antibody domain.

In some embodiments, in (B) said humanization of said sequence is by replacing at least one amino acid of said sequence by the corresponding amino acid of a human VH (variable heavy) domain.

In some embodiments, in (B) said humanization of said sequence is by (individually) replacing up to 25 amino acids of said sequence by the corresponding amino acids of a human VH domain.

As the skilled person understands, if the present disclosure refers to the replacement of an amino acid of a certain sequence/domain A by the "corresponding" amino acid of a certain sequence/domain B, this designates that said amino acid of sequence/domain A is replaced by the amino acid in sequence/domain B that in an alignment of the two sequences aligns with said amino acid of sequence/domain A.

In some embodiments, in (B) said humanization of said sequence is by replacing up to 20 amino acids of said sequence by the corresponding amino acids of a human VH domain.

In some embodiments, in (B) said humanization of said sequence is by replacing up to 15 amino acids of said sequence by the corresponding amino acids of a human VH domain.

In some embodiments, in (B) said humanization of said sequence is by replacing up to 10 amino acids of said sequence by the corresponding amino acids of a human VH domain.

In some embodiments, in (B) said humanization of said sequence is by replacing up to 5 amino acids of said sequence by the corresponding amino acids of a human VH domain.

In some embodiments, in (B) said humanization of said sequence is by replacing up to 3 amino acids of said sequence by the corresponding amino acids of a human VH domain.

In some embodiments, in (B) said humanization of said sequence is by replacing up to 2 amino acids of said sequence by the corresponding amino acids of a human VH domain.

In some embodiments, in (B) said humanization of said sequence is by replacing one amino acid of said sequence by the corresponding amino acid of a human VH domain.

In some embodiments, in (B) said humanization is within the framework regions of said VHH antibody domain and/or within the CDRs of said VHH antibody domain.

In some embodiments, in (B) said humanization is within the framework regions of said VHH antibody domain, but not within the CDRs of said VHH antibody domain.

In some embodiments, in (B) said humanization is within the CDRs of said VHH antibody domain, but not within the framework regions of said VHH antibody domain. In some embodiments, in (B) said humanization within the CDRs of said VHH antibody domain is within CDR1, CDR2 and/or CDR3.

In some embodiments, in (B) said humanization within the CDRs of said VHH antibody domain is within CDR1 and/or CDR2.

In some embodiments, in (B) said humanization within the CDRs of said VHH antibody domain is within CDR1.

In some embodiments, in (B) said humanization within the CDRs of said VHH antibody domain is within CDR2.

In some embodiments, in (B) said humanization within the CDRs of said VHH antibody domain is not within CDR3.

In some embodiments, in (C) the modification is the replacement, addition or deletion of up to 20 amino acids.

In some embodiments, in (C) the modification is the replacement, addition or deletion of up to 15 amino acids.

In some embodiments, in (C) the modification is the replacement, addition or deletion of up to 10 amino acids.

In some embodiments, in (C) the modification is the replacement, addition or deletion of up to 5 amino acids.

In some embodiments, in (C) the modification is the replacement, addition or deletion of up to 3 amino acids.

In some embodiments, in (C) the modification is the replacement, addition or deletion of up to 2 amino acids.

In some embodiments, in (C) the modification is the replacement, addition or deletion of one amino acid. In some embodiments, the modification in (C) comprises only the replacement, but not the addition or deletion of amino acids.

In some embodiments, in (A) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHHNKp46.11, VHH NKp46.12, VHHNKp46.13, VHHNKp46.14, VHHNKp46.15, VHH NKp46.16, VHH NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH NKp46.26, VHH NKp46.27, VHHNKp46.28, VHHNKp46.29, VHHNKp46.31 and VHHNKp46.34 as shown in the Table of CDRs.

In some embodiments, in (B) to (D)

(a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHHNKp46.1, VHHNKp46.2, VHHNKp46.3, VHHNKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29, VHH NKp46.31 and VHH NKp46.34 as shown in the Table of CDRs;

(b) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with modification, wherein the modification is that the sequence of at least one of CDR1, CDR2 and CDR3 is humanized;

- the replacement, addition or deletion of up to three amino acids in CDR1,

- the replacement, addition or deletion of up to three amino acids in CDR2 and/or - the replacement, addition or deletion of up to three amino acids in CDR3.

In some embodiments, said humanization of said CDR(s) is by replacing up to two amino acids in the sequence of CDR1 and/or CDR2 and up to one amino acid in the sequence of CDR3 by the corresponding amino acid of a human VH domain.

In some embodiments, said humanization of said CDR(s) is by replacing one amino acid in the sequence of said CDR by the corresponding amino acid of a human VH domain. In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to three amino acids in CDR1,

- the replacement, addition or deletion of up to two amino acids in CDR3.

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to three amino acids in CDR1,

- the replacement, addition or deletion of up to one amino acid in CDR3.

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to three amino acids in CDR1, and/or

- the replacement, addition or deletion of up to three amino acids in CDR2, wherein the sequence of CDR3 is unmodified.

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to two amino acids in CDR1,

- the replacement, addition or deletion of up to two amino acids in CDR2 and/or

- the replacement, addition or deletion of up to two amino acids in CDR3;

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to two amino acids in CDR1;

- the replacement, addition or deletion of up to one amino acid in CDR3.

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to two amino acids in CDR1 and/or

- the replacement, addition or deletion of up to two amino acids in CDR2; wherein the sequence of CDR3 is unmodified. As the skilled person is aware, "unmodified" mean unmodified compared to the sequence in the Table of CDRs.

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to two amino acids in CDR1, wherein the sequence of CDR2 and CDR3 is unmodified. In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to two amino acids in CDR2, wherein the sequence of CDR1 and CDR3 is unmodified.

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of up to one amino acid in CDR1;

- the replacement, addition or deletion of up to one amino acid in CDR2; and/or

- the replacement, addition or deletion of up to one amino acid in CDR3.

In some embodiments, the modification in (c) is

- the replacement, addition or deletion of one amino acid in CDR1 and/or

In some embodiments, the modification in (c) is

In some embodiments, the modification in (c) comprises only the replacement, but not the addition or deletion of amino acids.

In some embodiments, said compound comprises a VHH antibody domain (not only a fragment of a VHH antibody domain).

In some embodiments, said compound consists of a fragment of a VHH antibody domain (not a full-length VHH antibody domain).

In some embodiments, said fragment consists of at least 100 amino acids. In some embodiments, said fragment consists of at least 105 amino acids.

In some embodiments, said fragment consists of at least 110 amino acids.

In some embodiments, said fragment consists of at least 115 amino acids.

In some embodiments, said compound is capable of specifically binding to NKp46.

In some embodiments, the specific term that a certain compound, domain or fragment "is capable of specifically binding to NKp46" means that said compound, domain or fragment is capable of binding to NKp46 with an affinity of that is at least equal to the affinity with which VHH NKp46.31 (SEQ ID NO: 31) binds to NKp46 (or stronger). Such binding can be determined by in vitro binding experiments as described in Example 1 below (by biolayer interferometry).

In some embodiments, said VHH antibody domain or fragment thereof is capable of specifically binding to NKp46.

In some embodiments, said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a KD of IxlO'⁶ M or stronger.

In some embodiments, said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a KD of IxlO'⁷ M or stronger.

In some embodiments, said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a KD of 3.0x1 O'⁸ M or stronger.

In some embodiments, said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a KD of IxlO'⁸ M or stronger.

In some embodiments, said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a KD of IxlO'⁹ M or stronger. In some embodiments, said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_on of 1x10⁴ 1/Ms or higher.

In some embodiments, said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_on of 1x10⁵ 1/Ms or higher.

In some embodiments, said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_on of 5xl0⁵ 1/Ms or higher.

In some embodiments, said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_Off of 5xl0'³ 1/s or lower.

In some embodiments, said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_Off of IxlO'³ 1/s or lower.

In some embodiments, said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_Off of IxlO'⁴ 1/s or lower.

In some embodiments, said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_Off of IxlO'⁵ 1/s or lower.

In some embodiments, said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_Off of IxlO'⁶ 1/s or lower.

In some embodiments, said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_Off of IxlO'⁷ 1/s or lower.

In some embodiments, in (b) and (c) the VHH antibody domain or fragment thereof binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 10 weaker than the binding of the corresponding VHH antibody domain without modification.

In some embodiments, in (b) and (c) the VHH antibody domain or fragment thereof binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 5 weaker than the binding of the corresponding VHH antibody domain without modification. In some embodiments, in (b) and (c) the VHH antibody domain or fragment thereof binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 2 weaker than the binding of the corresponding VHH antibody domain without modification.

In some embodiments, in (b) and (c) the VHH antibody domain or fragment thereof binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 1.5 weaker than the binding of the corresponding VHH antibody domain without modification.

In some embodiments, in (b) and (c) the VHH antibody domain or fragment thereof binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 10 stronger than the binding of the corresponding VHH antibody domain without modification.

In some embodiments, in (b) and (c) the VHH antibody domain or fragment thereof binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 5 stronger than the binding of the corresponding VHH antibody domain without modification.

In some embodiments, in (b) and (c) the VHH antibody domain or fragment thereof binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 2 stronger than the binding of the corresponding VHH antibody domain without modification.

In some embodiments, in (b) and (c) the VHH antibody domain or fragment thereof binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 1.5 stronger than the binding of the corresponding VHH antibody domain without modification.

In some embodiments, in (B) and (C) the VHH antibody domain binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 10 weaker than the binding of the corresponding VHH antibody domain without modification.

In some embodiments, in (B) and (C) the VHH antibody domain binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 5 weaker than the binding of the corresponding VHH antibody domain without modification. In some embodiments, in (B) and (C) the VHH antibody domain binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 2 weaker than the binding of the corresponding VHH antibody domain without modification.

In some embodiments, in (B) and (C) the VHH antibody domain binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 1.5 weaker than the binding of the corresponding VHH antibody domain without modification.

In some embodiments, in (B) and (C) the VHH antibody domain binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 10 stronger than the binding of the corresponding VHH antibody domain without modification.

In some embodiments, in (B) and (C) the VHH antibody domain binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 5 stronger than the binding of the corresponding VHH antibody domain without modification.

In some embodiments, in (B) and (C) the VHH antibody domain binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 2 stronger than the binding of the corresponding VHH antibody domain without modification.

In some embodiments, in (B) and (C) the VHH antibody domain binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 1.5 stronger than the binding of the corresponding VHH antibody domain without modification.

In some embodiments, in (D) the affinity (KD value) of the binding of the VHH antibody domain to human NKp46 is by not more than a factor of 10 weaker than the affinity (KD value) of the binding to human NKp46 of a VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has the highest degree of sequence identity with the sequence of said VHH antibody domain of (D). The degree of sequence identity can be determined by sequence alignment.

In some embodiments, in (D) the affinity (KD value) of the binding of the VHH antibody domain to human NKp46 is by not more than a factor of 5 weaker than the affinity (KD value) of the binding to human NKp46 of a VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has the highest degree of sequence identity with the sequence of said VHH antibody domain of (D). The degree of sequence identity can be determined by sequence alignment.

In some embodiments, in (D) the affinity (KD value) of the binding of the VHH antibody domain to human NKp46 is by not more than a factor of 2 weaker than the affinity (KD value) of the binding to human NKp46 of a VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has the highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

In some embodiments, in (D) the affinity (KD value) of the binding of the VHH antibody domain to human NKp46 is by not more than a factor of 1.5 weaker than the affinity (KD value) of the binding to human NKp46 of a VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has the highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

In some embodiments, in (D) the affinity (KD value) of the binding of the VHH antibody domain to human NKp46 is by not more than a factor of 10 stronger than the affinity (KD value) of the binding to human NKp46 of a VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has the highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

In some embodiments, in (D) the affinity (KD value) of the binding of the VHH antibody domain to human NKp46 is by not more than a factor of 5 stronger than the affinity (KD value) of the binding to human NKp46 of a VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has the highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

In some embodiments, in (D) the affinity (KD value) of the binding of the VHH antibody domain to human NKp46 is by not more than a factor of 2 stronger than the affinity (KD value) of the binding to human NKp46 of a VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has the highest degree of sequence identity with the sequence of said VHH antibody domain of (D). In some embodiments, in (D) the affinity (KD value) of the binding of the VHH antibody domain to human NKp46 is by not more than a factor of 1.5 stronger than the affinity (KD value) of the binding to human NKp46 of a VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has the highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

In some embodiments, said KD value/said k_on rate/said k_Off rate is measured by kinetic measurements by biolayer interferometry at 25°C and 1000 rpm in KB Buffer (PBS + 0.1 % Tween-20 + 1% BSA).

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29 or VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 1.0E-08 M or stronger was observed for binding to NKp46.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.21, VHH NKp46.23, VHH NKp46.25, VHH NKp46.28, VHH NKp46.29 or VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 5.0E-09 M or stronger was observed for binding to NKp46.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.13, VHH NKp46.14, VHH NKp46.21, VHH NKp46.29 or VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 2.0E-09 M or stronger was observed for binding to NKp46.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.5, VHH NKp46.10 or VHH NKp46.29 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 1.0E-09 M or stronger was observed for binding to NKp46.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.10 or VHH NKp46.29 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 5.0E-10 M or stronger was observed for binding to NKp46.)

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.29 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 1.0E-11 M or stronger was observed for binding to NKp46.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.25, VHH NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29 or VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 1.0E+05 1/Ms or higher was observed for binding to NKp46.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.8, VHH NKp46.9, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.18, VHH NKp46.21, VHH NKp46.22, VHH NKp46.25, VHH NKp46.26, VHH NKp46.27 or VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 2.0E+05 1/Ms or higher was observed for binding to NKp46.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.1, VHH NKp46.4, VHH NKp46.5, VHH NKp46.8, VHH NKp46.9, VHH NKp46.12, VHH NKp46.13 or VHH NKp46.21 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 5.0E+05 1/Ms or higher was observed for binding to NKp46.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.1, VHH NKp46.4, VHH NKp46.5, VHHNKp46.9 or VHHNKp46.21 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 6.0E+05 1/Ms or higher was observed for binding to NKp46.)

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.21 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 7.0E+05 1/Ms or higher was observed for binding to NKp46.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5,

VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH

NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH

NKp46.16, VHH NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH

NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.26, VHH NKp46.27, VHH

NKp46.28, VHH NKp46.29, VHH NKp46.31 or VHH NKp46.34 shown in the Table of VHH

Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 5.0E-03 1/Ms or lower was observed for binding to

NKp46.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.14, VHH NKp46.20, VHH NKp46.23, VHH NKp46.24, VHH NKp46.28, VHH NKp46.29, VHH NKp46.31 or VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 1.0E-03 1/Ms or lower was observed for binding to NKp46.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.2, VHH NKp46.5, VHH NKp46.6, VHHNKp46.10, VHHNKp46.23, VHH NKp46.28 or VHH NKp46.29 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 5.0E-04 1/Ms or lower was observed for binding to NKp46.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.10 or VHH NKp46.29 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 1.0E-07 1/Ms or lower was observed for binding to NKp46.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.2, VHH NKp46.4, VHH NKp46.8, VHH NKp46.13, VHH NKp46.15, VHH NKp46.18, VHH NKp46.21 or VHH NKp46.25 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain belong to a specific epitope bin (epitope bin 1) with common characteristics.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.22 or VHH NKp46.26 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain are directed to an epitope that overlaps with epitope bin 1.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.2, VHH NKp46.4, VHH NKp46.8, VHH NKp46.13, VHH NKp46.15, VHH NKp46.18, VHH NKp46.21, VHH NKp46.22, VHH NKp46.25 or VHH NKp46.26 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain either belong to epitope bin 1 or are directed to an epitope that overlaps with epitope bin 1.) In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain belong to a specific epitope bin (epitope bin 2).)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.4, VHH NKp46.17, VHH NKp46.23, VHH NKp46.24, VHH NKp46.29 or VHH NKp46.31 shown in the Table of VHH Sequences. (As can be seen from Example 3, compounds with such a VHH antibody domain allowed to prepare compounds that showed strong killing activity in the assay of Fig. 2a.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.2, VHH NKp46.4, VHH NKp46.8, VHH NKp46.13, VHH NKp46.15, VHH NKp46.18, VHH NKp46.21, VHH NKp46.22, VHH NKp46.25, VHH NKp46.26 or VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen from Example 3, compounds with such a VHH antibody domain allowed to prepare compounds that showed strong killing activity in the assay of Fig. 2B and 3 A.)

In some embodiments, said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.2, VHH NKp46.18 or VHH NKp46.21 shown in the Table of VHH Sequences. (As can be seen from Example 3, Fig. 2B and 3A, compounds with such a VHH antibody domain allowed to prepare compounds that showed particularly strong killing activity.)

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.1 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.2 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.3 shown in the Table of VHH Sequences. In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.4 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.5 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.6 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.7 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.8 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.9 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.10 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.11 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.12 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.13 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.14 shown in the Table of VHH Sequences. In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.15 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.16 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.17 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.18 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.20 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.21 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.22 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.23 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.24 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.25 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.26 shown in the Table of VHH Sequences. In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.27 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.28 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.29 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.31 shown in the Table of VHH Sequences.

In some embodiments, said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.34 shown in the Table of VHH Sequences.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHHNKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29 and VHH NKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 1.0E-08 M or stronger was observed for binding to NKp46.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHHNKp46.6, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHHNKp46.11, VHHNKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.21, VHH NKp46.23, VHH NKp46.25, VHH NKp46.28, VHH NKp46.29 and VHH NKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 5.0E-09 M or stronger was observed for binding to NKp46.) In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.13, VHH NKp46.14, VHH NKp46.21, VHH NKp46.29 and VHH NKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 2.0E-09 M or stronger was observed for binding to NKp46.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.5, VHH NKp46.10 and VHH NKp46.29 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 1.0E-09 M or stronger was observed for binding to NKp46.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.10 and VHH NKp46.29 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 5.0E-10 M or stronger was observed for binding to NKp46.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.29 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 1.0E-11 M or stronger was observed for binding to NKp46.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.25, VHH NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29 and VHH NKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 1.0E+05 1/Ms or higher was observed for binding to NKp46.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.8, VHH NKp46.9, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.18, VHH NKp46.21, VHH NKp46.22, VHH NKp46.25, VHH NKp46.26, VHH NKp46.27 and VHH NKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 2.0E+05 1/Ms or higher was observed for binding to NKp46.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.4, VHH NKp46.5, VHH NKp46.8, VHH NKp46.9, VHH NKp46.12, VHH NKp46.13 and VHH NKp46.21 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 5.0E+05 1/Ms or higher was observed for binding to NKp46.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHHNKp46.1, VHHNKp46.4, VHHNKp46.5, VHH NKp46.9 and VHH NKp46.21 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 6.0E+05 1/Ms or higher was observed for binding to NKp46.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.21 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 7.0E+05 1/Ms or higher was observed for binding to NKp46.) In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHHNKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29, VHH NKp46.31 and VHH NKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 5.0E-03 1/Ms or lower was observed for binding to NKp46.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.14, VHH NKp46.20, VHH NKp46.23, VHH NKp46.24, VHH NKp46.28, VHH NKp46.29, VHH NKp46.31 and VHH NKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 1.0E-03 1/Ms or lower was observed for binding to NKp46.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.2, VHH NKp46.5, VHH NKp46.6, VHH NKp46.10, VHH NKp46.23, VHH NKp46.28 and VHH NKp46.29 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 5.0E-04 1/Ms or lower was observed for binding to NKp46.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.10 and VHH NKp46.29 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 1.0E-07 1/Ms or lower was observed for binding to NKp46.) In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.2, VHH NKp46.4, VHH NKp46.8, VHH NKp46.13, VHH NKp46.15, VHH NKp46.18, VHH NKp46.21 and VHH NKp46.25 as shown in the Table of CDRs. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain belong to a specific epitope bin (epitope bin 1) with common characteristics.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.22 and VHH NKp46.26 as shown in the Table of CDRs. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain are directed to an epitope that overlaps with epitope bin 1.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.2, VHH NKp46.4, VHH NKp46.8, VHH NKp46.13, VHH NKp46.15, VHH NKp46.18, VHH NKp46.21, VHH NKp46.22, VHH NKp46.25 and VHH NKp46.26 as shown in the Table of CDRs. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain either belong to epitope bin 1 or are directed to an epitope that overlaps with epitope bin 1.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain belong to a specific epitope bin (epitope bin 2).)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHHNKp46.4, VHHNKp46.17, VHHNKp46.23, VHHNKp46.24, VHH NKp46.29 and VHH NKp46.31 as shown in the Table of VHH Sequences. (As can be seen from Example 3, compounds with such a VHH antibody domain allowed to prepare compounds that showed strong killing activity in the assay of Fig. 2a.) In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.2, VHH NKp46.4, VHH NKp46.8, VHH NKp46.13, VHH NKp46.15, VHH NKp46.18, VHH NKp46.21, VHH NKp46.22, VHH NKp46.25, VHH NKp46.26 and VHH NKp46.34 as shown in the Table of VHH Sequences. (As can be seen from Example 3, compounds with such a VHH antibody domain allowed to prepare compounds that showed strong killing activity in the assay of Fig. 2B and 3 A.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.2, VHH NKp46.18 and VHH NKp46.21 as shown in the Table of VHH Sequences. (As can be seen from Example 3, Fig. 2B and 3 A, compounds with such a VHH antibody domain allowed to prepare compounds that showed particularly strong killing activity.)

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.1 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.2 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.3 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.4 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.5 as shown in the Table of CDRs. In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.6 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.7 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.8 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.9 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.10 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.l l as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.12 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.13 as shown in the Table of CDRs. In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.14 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.15 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.16 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.17 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.18 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.20 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.21 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.22 as shown in the Table of CDRs. In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.23 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.24 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.25 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.26 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.27 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.28 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.29 as shown in the Table of CDRs.

In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.31 as shown in the Table of CDRs. In some embodiments, in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.34 as shown in the Table of CDRs.

In some embodiments, said VHH antibody domain or fragment thereof competes with VHH NKp46.2 for binding to human NKp46.

In some embodiments, said VHH antibody domain or fragment thereof partially competes with VHH NKp46.2 for binding to human NKp46.

In some embodiments, said VHH antibody domain or fragment thereof does not compete with VHH NKp46.2 for binding to human NKp46.

In some embodiments, said VHH antibody domain or fragment thereof competes with VHH NKp46.34 for binding to human NKp46.

In some embodiments, said VHH antibody domain or fragment thereof partially competes with VHH NKp46.34 for binding to human NKp46.

In some embodiments, said VHH antibody domain or fragment thereof does not compete with VHH NKp46.34 for binding to human NKp46.

In some embodiments, said humanization is by germlining.

In some embodiments, said germlining involves (preferably consists of)

- identifying the human VH germline sequence that is closest to the sequence to be humanized with respect to its sequence similarity; and

- replacing amino acid(s) in the sequence to be humanized by the amino acid(s) at the corresponding sequence position(s) in the closest human VH germline sequence.

Sequence similarity and corresponding sequence positions can be determined by sequence alignment as described above. In Fig. 14, the most similar human germline sequence for the NKp46-specific VHH sequences described herein are indicated. In some embodiments, said compound with humanization binds to human NKp46 with an affinity (KD value) that is not weaker by a factor of more than 10 compared to the binding of a corresponding compound without humanization to human NKp46. As a skilled person understands, the term "corresponding compound without humanization" refers to a compound that differs only by the amino acid changes introduced in order to carry out said humanization, but that is otherwise identical.

In some embodiments, said compound with humanization binds to human NKp46 with an affinity (KD value) that is not weaker by a factor of more than 5 compared to the binding of a corresponding compound without humanization to human NKp46.

In some embodiments, said compound with humanization binds to human NKp46 with an affinity (KD value) that is not weaker by a factor of more than 2 compared to the binding of a corresponding compound without humanization to human NKp46.

In some embodiments, said compound with humanization binds to human NKp46 with an affinity (KD value) that is not weaker than the binding of a corresponding compound without humanization to human NKp46.

In some embodiments, said affinity is determined by KD measurement.

In some embodiments, said compound is a molecule.

In some embodiments, said compound comprises or is a protein.

By stating that the compound "comprises" a protein, the present disclosure designates that the compound includes a part within its chemical structure that is a protein. A compound that comprises a protein may or may not comprise a part that is not a protein.

In some embodiments, said compound is a protein.

By stating that the compound "is" a protein, the present disclosure designates that the compound consists only of protein and does not comprise a part that is not a protein. In some embodiments, said VHH antibody domain is derived from a Camelid antibody. In some embodiments, said VHH antibody domain is derived from a Llama antibody.

In some embodiments, said compound comprises a VHH antibody domain or fragment thereof according to (A), (B) or (C).

In some embodiments, said compound comprises a VHH antibody domain or fragment thereof according to (A), (B) or (D).

In some embodiments, said compound comprises a VHH antibody domain or fragment thereof according to (A), (C) or (D).

In some embodiments, said compound comprises a VHH antibody domain or fragment thereof according to (A) or (B).

In some embodiments, said compound comprises a VHH antibody domain or fragment thereof according to (A) or (C).

In some embodiments, said compound comprises a VHH antibody domain or fragment thereof according to (A) or (D).

In some embodiments, said compound comprises a VHH antibody domain or fragment thereof according to (A).

In some embodiments, said compound comprises a VHH antibody domain or fragment thereof according to (B).

In some embodiments, said VHH antibody domain or fragment thereof comprises complementarity determining regions according to (a) or (b).

In some embodiments, said VHH antibody domain or fragment thereof comprises complementarity determining regions according to (a) or (c). In some embodiments, said VHH antibody domain or fragment thereof comprises complementarity determining regions according to (a).

In some embodiments, said VHH antibody domain or fragment thereof comprises complementarity determining regions according to (b).

In some embodiments, said compound further comprises a targeting moiety.

As used herein, the term "targeting moiety" refers to a moiety (i.e. a molecular group or chemical structure) that is (typically covalently) associated with said compound and that binds a target site, wherein said binding allows to recruit the compound to said target site (e.g. a moiety that specifically binds to EGFR and thus targets the compound to cells expressing EGFR at their cell surface). The target site will typically be a biological molecule or a certain part of a biological molecule. An example of a targeting moiety is an antigen-binding antibody fragment that is covalently linked to an NKp46-binding VHH antibody domain to form a compound according to the present disclosure, wherein the antigen-binding fragment binds to a certain receptor present at the surface of a certain cell type (its antigen), and wherein binding of the antigen-binding fragment to this receptor results in recruitment of the compound to this cell type (e.g. the bispecific NKp46- and EGFR-binding molecule of Fig. 1 A).

Non-targeted drugs typically reach their site of action by whole-body distribution and passive diffusion. In contrast, targeted compounds are not distributed evenly across the whole body. Due to the interaction of targeting moiety with its target molecule, a compound including a targeting moiety is concentrated preferentially at its site target site. Therefore, e.g. therapeutic compounds with a targeting moiety require lower dosages to be therapeutically effective, thus improving the therapeutic window.

In some embodiments, all components of said compound are covalently linked.

In some embodiments, said targeting moiety is a molecular group that specifically binds to a target molecule or fragment thereof.

In some embodiments, said target molecule is a receptor at the surface of a cell. In some embodiments, said target molecule is an antigen that is present on the surface of a target cell.

As used herein, a "target molecule that is present on the surface of a target cell" is a molecule that is present on the surface of the target cell in such a manner that it is accessible from the extracellular environment (i.e. e.g. an antibody can bind to it from the extracellular environment). For example, CD8 is a transmembrane protein of cytotoxic T cells, and its extracellular domain is accessible for antibodies directed against the extracellular domain of CD8 from the extracellular environment. Thus, in the sense of the present disclosure, CD8 is a target molecule that is present on the surface of cytotoxic T cells.

A targeting moiety that "binds" a target molecule of interest is a targeting moiety that is capable of binding that target molecule with sufficient affinity such that the targeting moiety is useful in targeting the compound to a cell expressing the target molecule.

If the present disclosure refers to a first molecule/molecular group (e.g. an antib ody/antibody component) "specifically binding"/that "specifically binds" to a second molecule/molecular group (e.g. an antigen of interest), this means that the first molecule/molecular group (in this example the antibody) binds to said second molecule/molecular group (in this example the antigen of interest) with an affinity that is at least ten-fold greater than its affinity for other molecules/molecular groups, in particular other molecule/molecular group in the human body (in this example at least ten-fold greater than its affinity for binding to non-specific antigens (e.g., BSA, casein) other than said antigen of interest (or closely related antigens)). In a preferred embodiment, a first molecule/molecular group (e.g. an antib ody/antibody component) that "specifically binds" to a second molecule/molecular group (e.g. an antigen of interest) binds to said antigen with an affinity that is at least 100-fold greater than its affinity for other molecules/molecular groups, in particular other molecule/molecular group in the human body (in this example at least 100-fold greater than its affinity for binding to non-specific antigens other than said antigen of interest (or closely related antigens)). Typically said binding will be determined under physiological conditions. A first molecule/molecular group that "specifically binds" to a second molecule/molecular group may bind to that second molecule/molecular group with a KD of 1 * 10'⁷ M or stronger. In some embodiments, said targeting moiety is a protein, a peptide, a peptide mimetic, a nucleic acid, an oligonucleotide or a small molecule.

As used herein, the term "peptide mimetic" refers to a peptide-like chain which is designed to mimic a peptide. An example of a peptide mimetic is a D-peptide mimetic containing a D- amino acid, but is not limited thereto.

As used herein, a "small molecule", is a molecule with a molecular weight < 1000 Da.

In some embodiments, said targeting moiety comprises or is a protein.

In some embodiments, said targeting moiety comprises a protein.

In some embodiments, said targeting moiety is a protein.

In some embodiments, said targeting moiety is a protein ligand that specifically binds to a receptor at the surface of a cell.

In some embodiments, said targeting moiety is an antibody or an antigen-binding fragment thereof.

In some embodiments, said targeting moiety is capable of specifically binding to a tumor- associated antigen.

In some embodiments, said compound is a bispecific antibody. If the present disclosure states that the compound is a bispecific antibody, that does not exclude the possibility that said bispecific antibody is linked to further domains or moieties.

In some embodiments, said compound is a bispecific antibody prepared by the SEED (strandexchange engineered domain) technology.

The term "bispecific antibody", as used in the present disclosure, refers to an antibody that is capable of specifically binding to two different epitopes at the same time. The terms "epitope" or "antigenic determinant" are used interchangeably herein and refer to the portion of an antigen that is recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

The two different epitopes to which a bispecific antibody binds can be from the same antigen or from two different antigens. Preferably, the two epitopes are from two different antigens. Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using the co-expression of two immunoglobulin heavy chain/light chain pairs (see e.g. Milstein et al., Nature (1983), vol. 305, p. 537-539). Alternatively, bispecific antibodies can be prepared using chemical linkage (see e.g. Brennan et al., Science (1985), vol. 229, p. 81). A bispecific antibody can also for example be prepared by the SEED technology (an approach for generation of bispecific antibodies in which structurally related sequences within the conserved CH3 domains of human IgA and IgG are exchanged to form two asymmetric but complementary domains, see WO 2016/087650). See the Examples section for further details. A bispecific antibody prepared by the SEED technology is herein referred to as "SEEDbody".

In some embodiments, one binding site of said bispecific antibody is formed by said VHH antibody domain or fragment thereof and one binding site of said bispecific antibody is formed by said targeting moiety.

In some embodiments, said antigen-binding fragment is selected from the group consisting of a Fab, a Fab', a (Fab')2, a Fv, a scFv, a diabody and a VHH.

"Fab" fragments are obtained by papain digestion of an antibody, which produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.

"F(ab')2" fragments are obtained by pepsin treatment of an antibody, which yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen.

"Fab¹ " fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.

"Fv" is the minimum antibody fragment which contains a complete antigen-recognition and - binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

"Single-chain Fv", also abbreviated as "scFv", are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of the scFv, see Pluckthun, in: The Pharmacology of Monoclonal Antibodies, vol. 113 (1994), editors Rosenburg and Moore, Springer-Verlag (New York), p. 269-315. The term "diabody" refers to a small antibody fragment prepared by constructing scFv fragments (see preceding paragraph) with short linkers (about 5-25 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" scFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in greater detail in, for example, EP 0404097; WO 93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA (1993), vol. 90, p. 6444-6448.

In some embodiments, said antigen-binding fragment is selected from the group consisting of a Fab, a Fab', a (Fab')2 and a Fv.

In some embodiments, said antigen-binding fragment is a Fab.

In some embodiments, said antigen-binding fragment is selected from the group consisting of a scFv, a diabody and a VHH.

In some embodiments, said antigen-binding fragment is an antigen-binding fragment of an antibody with the SEED (strand-exchange engineered domain) format.

In some embodiments, said targeting moiety is capable of specifically binding to an antigen that is present on the surface of a target cell.

In some embodiments, said antibody is an antibody against an antigen that is present on the surface of a target cell.

In some embodiments, said antigen-binding fragment is an antigen-binding fragment of an antibody against an antigen that is present on the surface of a target cell.

An antibody/antigen-binding fragment "against" a certain antigen is an antibody/antigen- binding fragment with an antigen-binding site that binds to said antigen. If an antibody/antigen- binding fragment binds to an antigen can e.g. be determined by testing in an immunofluorescence experiment with cultured cells whether the antibody binds to cells that express the antigen at their cell surface. In some embodiments, said antigen that is present on the surface of said target cell is more abundant on the surface of said target cell than on the surface of other cell types.

The abundance of a surface antigen on a cell type can be determined by standard methods known to a skilled person, e.g. flow cytometry (e.g. by exposing cell of said cell type to the antibody of interest, subsequently staining with a fluorescently labelled secondary antibody directed against the antibody of interest, and detection of fluorescent label by flow cytometry).

In some embodiments, said antigen that is present on the surface of said target cell is present on the surface of said target cell, but substantially not on the surface of other cell types.

As used herein, an antigen that is "present on the surface of said target cell, but substantially not on the surface of other cell types" is sufficiently abundant at the surface of the target cell to allow for recruitment of a compound with a targeting moiety (an antibody or antigen-binding fragment thereof) against said antigen under physiological conditions. In contrast, abundance of said antigen at the surface of other cell types is so low that recruitment of said compound under physiological conditions is barely above background binding.

In some embodiments, said antigen that is present on the surface of said target cell is present on the surface of said target cell, but not on the surface of other cell types.

As used herein, an antigen that is "present on the surface of said target cell, but not on the surface of other cell types" is sufficiently abundant at the surface of the target cell to allow for recruitment of a compound with a targeting moiety (an antibody or antigen-binding fragment thereof) against said antigen under physiological conditions. In contrast, abundance of said antigen at the surface of other cell types is so low that recruitment of said compound under physiological conditions is not above background binding.

In some embodiments, said binding of said targeting moiety to said antigen that is present on the surface of said target cell allows to recruit the compound specifically to said target cell.

The term "allows to recruit the antibody-drug conjugate specifically to said target cell" means that the compound is recruited to said target cell under physiological conditions with an efficiency that is at least 10 times higher, preferably at least 100 times higher, than the recruitment to other cell types (i.e. to other cell types to which said compound may be exposed in the body during administration of said compound).

In some embodiments, said antigen that is present on the surface of said target cell is a tumor- associated antigen.

As used herein, a "tumor-associated antigen" is, in its broadest sense, an antigen that allows recruitment of an ADC to the site of a tumor, such that a therapeutic action or diagnostic (e.g. labelling of the tumor site) can be achieved. The tumor-associated antigen may either be an antigen that is present on the surface of the tumor cells or an antigen associated with the tumor microenvironment.

Sources for information on cell surface expression and methods to identify and verify tumor- associated antigens are known to a skilled person and described in the literature (see e.g. Bomstein, AAPS J. (2015), vol. 17(3), p. 525-534; Hong et al., BMC Syst Biol. (2018), vol. 12 (Suppl 2), p. 17; Immune Epitope Database and Analysis Resource (https://www.iedb.org); Cancer Cell Line Encyclopedia (https://portals.broadinstitute.org/ccle); OASIS Database (http ://oasi s-genomics . org/)) .

In preferred embodiments, said tumor-associated antigen is an antigen that is present on the surface of a tumor cell. In these embodiments, the term "tumor-associated antigen" indicates an antigen that is present at the cell surface of a tumor cell and allows for distinction of the tumor cell over other cell types. A tumor-associated antigen may be part of a molecule (e.g. a protein) that is expressed by a tumor cell and accessible from the extracellular environment. A tumor- associated antigen may differ (i.e. qualitatively differ) from its counterpart in corresponding non-tumor cells (e.g., where the molecule is a protein by one or more amino acid residues). Alternatively, the tumor-associated antigen may be identical to its counterpart in corresponding non-tumor cells, but present on the surface of the tumor cells at a higher level than on the surface of corresponding non-tumor cells. For example, the tumor-associated antigen may be present only on the surface of the tumor cells, but not on the surface of non-tumor cells, or the tumor- associated antigen may be present on the surface of tumor cells at a higher level (e.g. at least 5- fold higher, preferably at least 100-fold higher) than on the surface of non-tumor cells. In an embodiment, the tumor-associated antigen is present on the surface of tumor cells at a level that is at least 1000-fold higher than on the surface of non-tumor cells.

In some embodiments, said tumor-associated antigen is an antigen that is present on the surface of a tumor cell.

In some embodiments, said tumor-associated antigen is EGFR (epidermal growth factor receptor).

In some embodiments, said compound is an NK cell engager (NKCE).

In some embodiments, said compound is a bispecific or multispecific molecule. In some embodiments, said compound is a multispecific molecule. In some embodiments, said compound is a bispecific or multispecific molecule that binds via its VHH antibody domain or fragment thereof to NKp46 and via its targeting domain to a tumor-associated antigen.

In some embodiments, said compound comprises an antibody Fc region.

As used herein, the term "antibody Fc region" refers to the portion of a native immunoglobulin formed by the Fc domains of its two heavy chains (which includes a heavy chain constant region 1 (CHI), a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) of an immunoglobulin, but does not include variable regions of the heavy and light chains and a light chain constant region 1 (CL1) of an immunoglobulin). A native Fc region is homodimeric. In some embodiments, the term includes variant Fc regions with one or more alterations relative to a native Fc region. An Fc region may be altered by amino acid substitutions, additions and/or deletions, linkage of additional moieties, and/or alteration of the native glycans. The term encompasses Fc regions wherein each of the constituent Fc domains is different. Examples of heterodimeric Fc regions include, without limitation, Fc regions made using the "knobs into holes" technology as described in, for example US Patent No. 8,216,805 or by the SEED technology as described in WO 2016/087650. In some embodiments, said compound comprises an antibody Fc region competent in Fc receptor binding.

An antibody Fc region is "competent in FC receptor binding" if said antibody Fc region is capable of binding to at least one of the Fc receptors (FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors).

In some embodiments, said compound comprises an antibody Fc region that is not competent in Fc receptor binding.

In some embodiments, said compound does not comprise an effector-competent antibody Fc region.

An "effector-competent" Fc region is an Fc region having the functional ability to bind proteins and/or cells of the immune system and mediate biological effects normally induced following the binding of an antibody to a corresponding antigen. Such biological effects include e.g. the ability to bind a complement protein (e.g. Clq), resulting in activation of the classical complement system leading to the opsonisation and lysis of cell pathogens (complementdependent cytotoxicity, CDCC). Other biological effects are endocytosis of immune complexes, engulfment and destruction of antibody-coated particles or microorganisms (also called antibody-dependent phagocytosis, or ADCP), clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, regulation of immune system cell activation or control of immunoglobulin production.

In some embodiments, said compound comprises an effector-competent antibody Fc region.

In some embodiments, said compound does not comprise an antibody Fc region capable of inducing ADCC (antibody-dependent cellular cytotoxicity).

In some embodiments, said compound comprises an antibody Fc region capable of inducing ADCC. In some embodiments, said compound comprises an antibody Fc region that is not capable of inducing ADCC.

In some embodiments, said compound is capable of inducing both FcyRIIIa signalling and positive (i.e. NK cell activating) NKp46 signalling.

In some embodiments, said compound is capable of specifically binding toNKp46 on NK cells.

In some embodiments, said compound is capable of activating NK cells by binding to NKp46 on said NK cells.

Whether a compound or domain is, upon binding to NKp46 on NK cells, capable of activating NK cells can be determined as described in Example 1, section "NK cell activation assay". If this activation occurs upon binding to NKp46 on NK cells can for example be determined by carrying out a control experiment with NK cells in which NKp46 has been blocked by a competitor molecule that binds to NKp46 such that the protein domain cannot get access to NKp46 on the NK cells.

Preferably, binding to NKp46 and activation of NK cells is assessed with said VHH antibody domain in the context of the complete compound.

In some embodiments, binding of said compound to NKp46 on NK cells activates said NK cells.

In some embodiments, said compound is an agonist of NKp46.

In some embodiments, said compound is, upon binding of said VHH antibody domain or fragment thereof to NKp46 on NK cells, capable of activating NK cells.

In some embodiments, activation of NK cells is measured by measuring expression of the activation marker CD69 by flow cytometry. In some embodiments, said compound comprises one NKp46-specific VHH domain and one targeting moiety, wherein said targeting moiety is a Fab.

In some embodiments, said compound comprises two NKp46-specific VHH domains and one targeting moiety, wherein said targeting moiety is a Fab.

In some embodiments, said compound comprises two NKp46-specific VHH domains and two targeting moieties, wherein said targeting moieties are Fabs.

In some embodiments, said compound comprises one NKp46-specific VHH domain and two targeting moieties, wherein said targeting moieties are Fabs.

In some embodiments, said Fab is/said Fabs are located at the N-terminus of the molecule.

In some embodiments, said NKp46-specific VHH domain is/said NKp46-specific VHH domains are located at the N-terminus of the compound.

In some embodiments, said NKp46-specific VHH domain is/said NKp46-specific VHH domains are located at the C-terminus of the compound.

In some embodiments, said compounds shows cytotoxic activity. Such activity can for example be determined by a protocol as exemplified in Example 1, section "Tumor cell killing assays" below.

In some embodiments, said compound shows cytotoxic activity in a ⁵¹Cr release assay.

In some embodiments, said ⁵¹Cr release assay is carried out as a 4 h ⁵¹Cr release assay, wherein human PBMCs are used as effector cells at effector-to-target cell (E:T) ratios of 80: 1, and wherein a higher percent lysis indicates improved cytotoxic activity.

In some embodiments, said ⁵¹Cr release assay is carried out as described in Repp et al., 2011. As used herein, the reference "Repp et al., 2011" refers to the publication R. Repp et al., "Combined Fc-protein- and Fc-gly co-engineering of scFv-Fc fusion proteins synergistically enhances CD 16a binding but does not further enhance NK-cell mediated ADCC," Journal of Immunological Methods (2011), vol. 373, p. 67-78.

Specifically, tumor cell killings assays by 51Cr release assay can be performed as follows: Preparation of PBMCs from healthy donors can be performed as described in Repp et al., 2011. NK cells can be isolated by negative selection using an NK cell isolation kit (e.g. Miltenyi Biotech) and maintained overnight at a density of 2xl0⁶ cells/ml in R10+ medium. Cytotoxicity can be analyzed in standard 4 h ⁵¹Cr release assays performed in 96-well microtiter plates in a total volume of 200 pl as described in Repp et al., 2011. Human PBMCs or purified NK cells can be used as effector cells at effector-to-target cell (E:T) ratios of 80: 1 and 10: 1, respectively.

Cytokine release assays for the quantification of IFN-y and TNF-a released by NK cells can be performed using human cytokine HTRF kits (e.g. cisbio) as described in Pekar et al., 2021. In brief, 2.500 viable EGFR-positive A431 cells per well or EGFR-negative CHO cells are seeded in 384 clear bottom microtiter plates and incubated for 3 h. 12.500 viable NK cells are added after overnight incubation in complete medium containing lOO U/ml recombinant human interleukin-2 (R&D systems), resulting in an effector cell to target cell (E:T) ratio of 5: 1. Compounds to be tested (e.g. SEEDbodies) are added to a final concentration of 50 nM. As controls, tumor cells only as well as NK cells cultivated with tumor cells in absence of NK cell engagers are utilized. After 24 h incubation, cells are sedimented by centrifugation and cytokine containing supernatants are further processed according to manufacturer’s instructions. Assay plates are measured. HTRF optical entity using excitation at 337 nM and emission at 620 nM as well as 665 nM can be utilized. Analyses and fitting of resulting data can e.g. be carried out with MARS software (v.3.32, BMG) enabling a 4 Parameter Logistic (4PL 1/y²) model fitting of the standard curve following kit manufacturer’s instructions.

In some embodiments, binding of said compound to NKp46 on NK cells results in the release of interferon-y (IFN-y).

In some embodiments, said release of IFN-y is measured as follows: isolated human NK cells are incubated overnight in medium containing 100 U/ml recombinant human interleukin-2, in different wells A431 cells are seeded and incubated for 3 h, the compound is added to a final concentration of 85 nM followed by addition of NK cells at an E:T ratio of 5: 1, human IFN-y is analyzed in the culture supernatant after 24 h by ELISA.

In some embodiments, binding of said compound to NKp46 on NK cells results in the release of tumor necrosis factor-a (TNF-a).

In some embodiments, said release of TNF-a is measured as follows: isolated human NK cells are incubated overnight in medium containing 100 U/ml recombinant human interleukin-2, in different wells A431 cells are seeded and incubated for 3 h, the compound is added to a final concentration of 85 nM followed by addition of NK cells at an E:T ratio of 5: 1, human TNF-a is analyzed in the culture supernatant after 24 h by ELISA.

According to another aspect, the present disclosure relates to a pharmaceutical composition comprising the compound according to any one of the aspects or embodiments described above.

Methods for preparing pharmaceutical compositions are known to a skilled person in the art (Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), Pharmaceutical Press).

In some embodiments, said pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent and/or excipient.

The term "pharmaceutically acceptable" designates that said carrier, diluent or excipient is a non-toxic, inert material that is compatible with the other ingredients of the pharmaceutical composition and not harmful to the patient that the pharmaceutical composition is administered to, such that it can be used in a pharmaceutical product. Substances suitable as carriers, diluents or excipients in pharmaceutical compositions are known to a skilled person in the art (Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), Pharmaceutical Press). The pharmaceutical composition may further include e.g. additional adjuvants, antioxidants, buffering agents, bulking agents, colorants, emulsifiers, fillers, flavoring agents, preservatives, stabilizers, suspending agents and/or other customary pharmaceutical auxiliaries.

In some embodiments, said pharmaceutical composition further includes at least one additional adjuvant, antioxidant, buffering agent, bulking agent, colorant, emulsifier, filler, flavoring agent, preservative, stabilizer, suspending agent and/or other customary pharmaceutical auxiliary.

According to another aspect, the present disclosure relates to a compound according to any of the aspects or embodiments described above or a pharmaceutical composition according to any of the aspects or embodiments described above for use as a medicament.

According to another aspect, the present disclosure relates to a compound according to any of the aspects or embodiments described above or a pharmaceutical composition according to any of the aspects or embodiments described above for use in the treatment of cancer.

According to another aspect, the present disclosure relates to a compound according to any of the aspects or embodiments described above or a pharmaceutical composition according to any of the aspects or embodiments described above for use in the treatment of a malignant tumor.

In some embodiments, said compound/said pharmaceutical composition is for use in the treatment of a human.

The production of medicaments containing the compound of the present disclosure according or a pharmaceutical composition according to the present disclosure can be performed according to well-known pharmaceutical methods. Further details on techniques for formulation and administration may be found e.g. in Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), Pharmaceutical Press.

As used herein, "treatment" of a disease and "treating" a disease refers to the process of providing a subject with a pharmaceutical treatment, e.g., the administration of a drug, such that said disease is alleviated, reduced, minimized, halted or even healed, and/or such that the chances of a relapse into the disease are reduced or a relapse into the disease is even prevented.

The use of compounds in the treatment of diseases is known to a skilled person in the art (see e.g. Coats et al., Clinical Cancer Research (2019), vol. 25(18), p. 5441-5448; Rudra, Bioconjugate Chemistry (2020), vol. 31(3), p. 462-473). Thus, the skilled person is aware that the components of the compound, in particular the targeting moiety, must be selected appropriately in order to allow for successful treatment. For example, for treatment of a specific cancer, the targeting moiety of the compound must be selected such that binding of the targeting moiety to its target site directs the compound to said cancer (e.g. by using an antibody component against a tumor-associated antigen that is specifically found on the surface of the cancer cells). Cytotoxic effects will then be achieved by the affinity-matured variant B7-H6 sequence included in the compound. In addition, a payload may be included in the compound such that an additional desired treatment effect is achieved. For example, for the treatment of a cancer, a cytotoxic drug may be included in addition.

In another aspect, the present disclosure relates to a method for treating a disease in a patient in need thereof, comprising the step of administering to said patient a therapeutically effective amount of the compound according to any of the aspects or embodiments described above or the pharmaceutical composition according to any of the aspects or embodiments described above.

By "therapeutically effective amount" is meant the amount of an agent required to ameliorate the symptoms of a disease. The effective amount of active agent(s) (e.g., a compound according to the present disclosure) used for therapeutic treatment of a disease according to the present disclosure varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as a "therapeutically effective" amount.

The term "patient", as used herein, refers to a mammal (such as a human, rat, mouse, monkey, pig, goat, cow, horse, dog or cat). Preferably, the patient is a human.

In some embodiments, said disease is cancer.

In some embodiments, said disease is a malignant tumor.

As used herein, the term "cancer" refers to a malignant neoplasm. Cancer can include a hematological cancer or a solid tumor. For example, the cancer can be a leukemia (e.g., acute myeloid leukemia (AML), acute monocytic leukemia, promyelocytic leukemia, eosinophilic leukaemia, acute lymphoblastic leukemia (ALL) such as acute B lymphoblastic leukemia (B- ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL)) or lymphoma (e.g., non-Hodgkin lymphoma), myelodysplastic syndrome (MDS)" melanoma, lung cancer (e.g., non-small cell lung cancer; NSCLC), ovarian cancer, endometrial cancer, peritoneal cancer, pancreatic cancer, breast cancer, prostate cancer, squamous cell carcinoma of the head and neck, or cervical cancer. Preferably, in the present disclosure the term "cancer" refers to a solid malignant tumor.

In some embodiments, said patient is a human.

In another aspect, the present disclosure relates to the use of the compound according to any of the aspects or embodiments described above or of the pharmaceutical composition according to any of the aspects or embodiments described above for the manufacture of a medicament for the treatment of cancer.

In another aspect, the present disclosure relates to the use of the compound according to any of the aspects or embodiments described above or of the pharmaceutical composition according to any of the aspects or embodiments described above for the manufacture of a medicament for the treatment of a malignant tumor.

In some embodiments, said medicament is prepared for administration to a human.

The following embodiments relate to any of the compounds or pharmaceutical compositions for use in medical treatment, methods for treating a disease in a patient in need thereof, uses for the manufacture of a medicament, or any of their embodiments described above.

In some embodiments, said cancer or malignant tumor is a human disease.

In some embodiments, said compound binds to NKp46 with a KD of 5 * 10'⁶ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA).

In some embodiments, said compound binds to NKp46 with a KD of 1 x 10'⁶ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) of NKp46 in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA). In some embodiments, said compound binds to NKp46 with a KD of 5 * 10'⁷ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA).

In some embodiments, said compound binds to NKp46 with a KD of 1 x 10'⁷ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA).

In some embodiments, said compound binds to NKp46 with a KD of 5 * 10'⁸ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA).

In some embodiments, said compound binds to NKp46 with a KD of 1 x 10'⁸ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA).

In some embodiments, said compound binds to NKp46 with a KD of 5 x 10'⁹ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA).

In some embodiments, said compound binds to NKp46 with a KD of 1 x 10'⁹ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA).

Also disclosed with regard to the above-described subject matter is the following:

[1] A compound comprising a VHH antibody domain or a fragment thereof, wherein

(a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH

NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH

NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29, VHH

NKp46.31 and VHH NKp46.34 as shown in the Table of CDRs below;

(c) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with modification, wherein the modification is the replacement, addition or deletion of up to three amino acids in CDR1, the replacement, addition or deletion of up to three amino acids in CDR2 and/or the replacement, addition or deletion of up to three amino acids in CDR3;

Table of CDRs:

[2] The compound of item [1], wherein the modification in (b) is that the sequence of CDR1 and/or CDR2, but not the sequence of CDR3 is humanized.

[3] The compound of any one of items [1] or [2], wherein the modification in (b) is that the sequence of CDR1 is humanized, but not the sequence of CDR2 and CDR3.

[4] The compound of any one of items [1] or [2], wherein the modification in (b) is that the sequence of CDR2 is humanized, but not the sequence of CDR1 and CDR3.

[5] The compound of any one of items [1] to [4], wherein the modification in (b) is that the sequence of one, but not more than one of CDR1, CDR2 and CDR3 is humanized.

[6] The compound of any one of items [1] to [5], wherein said humanization of said CDR(s) is by replacing at least one amino acid in the sequence of said CDR by the corresponding amino acid of a human VH domain.

[7] The compound of any one of items [1] to [6], wherein said humanization of said CDR(s) is by replacing up to three amino acids in the sequence of said CDR by the corresponding amino acid of a human VH domain. [8] The compound of any one of items [1] to [6], wherein said humanization of said CDR(s) is by replacing up to three amino acids in the sequence of CDR1 and/or CDR2 and up to one amino acid in the sequence of CDR3 by the corresponding amino acid of a human VH domain.

[9] The compound of any one of items [1] to [6], wherein said humanization of said CDR(s) is by replacing up to two amino acids in the sequence of said CDR by the corresponding amino acid of a human VH domain.

[10] The compound of any one of items [1] to [6], wherein said humanization of said CDR(s) is by replacing up to two amino acids in the sequence of CDR1 and/or CDR2 and up to one amino acid in the sequence of CDR3 by the corresponding amino acid of a human VH domain.

[11] The compound of any one of items [1] to [10], wherein said humanization of said CDR(s) is by replacing one amino acid in the sequence of said CDR by the corresponding amino acid of a human VH domain.

[12] The compound of any one of items [1] to [11], wherein the modification in (c) is the replacement, addition or deletion of up to three amino acids in CDR1, the replacement, addition or deletion of up to three amino acids in CDR2 and/or the replacement, addition or deletion of up to one amino acid in CDR3.

[13] The compound of any one of items [1] to [11], wherein the modification in (c) is the replacement, addition or deletion of up to two amino acids in CDR1, the replacement, addition or deletion of up to two amino acids in CDR2 and/or the replacement, addition or deletion of up to two amino acids in CDR3;

[14] The compound of any one of items [1] to [11], wherein the modification in (c) is the replacement, addition or deletion of up to two amino acids in CDR1; the replacement, addition or deletion of up to two amino acids in CDR2; and/or the replacement, addition or deletion of up to one amino acid in CDR3.

[15] The compound of any one of items [1] to [11], wherein the modification in (c) is the replacement, addition or deletion of up to two amino acids in CDR1 and/or the replacement, addition or deletion of up to two amino acids in CDR2; wherein the sequence of CDR3 is unmodified.

[16] The compound of any one of items [1] to [11], wherein the modification in (c) is the replacement, addition or deletion of up to two amino acids in CDR1, wherein the sequence of CDR2 and CDR3 is unmodified.

[17] The compound of any one of items [1] to [11], wherein the modification in (c) is the replacement, addition or deletion of up to two amino acids in CDR2, wherein the sequence of CDR1 and CDR3 is unmodified.

[18] The compound of any one of items [1] to [11], wherein the modification in (c) is the replacement, addition or deletion of up to one amino acid in CDR1; the replacement, addition or deletion of up to one amino acid in CDR2; and/or the replacement, addition or deletion of up to one amino acid in CDR3.

[19] The compound of any one of items [1] to [11], wherein the modification in (c) is the replacement, addition or deletion of one amino acid in CDR1 and/or the replacement, addition or deletion of one amino acid in CDR2; wherein the sequence of CDR3 is unmodified.

[20] The compound of any one of items [1] to [11], wherein the modification in (c) is the replacement, addition or deletion of one amino acid in CDR1, wherein the sequence of CDR2 and CDR3 is unmodified.

[21] The compound of any one of items [1] to [11], wherein the modification in (c) is the replacement, addition or deletion of one amino acid in CDR2, wherein the sequence of CDR1 and CDR3 is unmodified. [22] The compound of any one of items [1] to [21], wherein the modification in (c) comprises only the replacement, but not the addition or deletion of amino acids.

[23] A compound comprising a VHH antibody domain or a fragment thereof, wherein

(A) said VHH antibody domain comprises the VHH sequence of a VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH

NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH

NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29, VHH

NKp46.31 and VHH NKp46.34 as shown in the Table of VHH Sequences below;

Table of VHH Sequences:

[24] A compound comprising a VHH antibody domain or a fragment thereof, wherein

(A) said VHH antibody domain consists of the VHH sequence of a VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH

NKp46.8, VHH NKp46.9, VHHNKp46.10, VHH NKp46.11, VHHNKp46.12,

VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH

NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH

NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH

NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29, VHH

NKp46.31 and VHHNKp46.34 as shown in the Table of VHH Sequences;

(B) said VHH antibody domain consists of a VHH sequence as defined in (A) with modification, wherein the modification is that said sequence is humanized; (C) said VHH antibody domain consists of a VHH sequence as defined in (A) with modification, wherein the modification is the replacement, addition or deletion of up to 25 amino acids; or

[25] The compound of any one of items [23] or [24], wherein said fragment of said VHH antibody domain comprises at least 75% of the amino acids of the sequence of said VHH antibody domain.

[26] The compound of any one of items [23] or [24], wherein said fragment of said VHH antibody domain comprises at least 80% of the amino acids of the sequence of said VHH antibody domain.

[27] The compound of any one of items [23] or [24], wherein said fragment of said VHH antibody domain comprises at least 85% of the amino acids of the sequence of said VHH antibody domain.

[28] The compound of any one of items [23] or [24], wherein said fragment of said VHH antibody domain comprises at least 90% of the amino acids of the sequence of said VHH antibody domain.

[29] The compound of any one of items [23] or [24], wherein said fragment of said VHH antibody domain comprises at least 95% of the amino acids of the sequence of said VHH antibody domain.

[30] The compound of any one of items [23] or [24], wherein said fragment of said VHH antibody domain comprises at least 98% of the amino acids of the sequence of said VHH antibody domain.

[31] The compound of any one of items [23] or [24], wherein said fragment of said VHH antibody domain comprises at least 99% of the amino acids of the sequence of said VHH antibody domain. [32] The compound of any one of items [23] to [31], wherein said fragment of said VHH antibody domain comprises complementarity determining regions CDR1, CDR2 and CDR3.

[33] The compound of any one of items [23] to [32], wherein said fragment of said VHH antibody domain comprises at least the sequence from the N-terminus of CDR1 to the C-terminus of CDR3 of said VHH antibody domain.

[34] The compound of any one of items [23] to [33], wherein in (A) said fragment of said VHH antibody domain comprises all the complementarity determining regions (CDRs) of said VHH antibody domain.

[35] The compound of any one of items [23] to [34], wherein in (B) said humanization of said sequence is by replacing at least one amino acid of said sequence by the corresponding amino acid of a human VH (variable heavy) domain.

[36] The compound of any one of items [23] to [35], wherein in (B) said humanization of said sequence is by replacing up to 25 amino acids of said sequence by the corresponding amino acids of a human VH domain.

[37] The compound of any one of items [23] to [35], wherein in (B) said humanization of said sequence is by replacing up to 20 amino acids of said sequence by the corresponding amino acids of a human VH domain.

[38] The compound of any one of items [23] to [35], wherein in (B) said humanization of said sequence is by replacing up to 15 amino acids of said sequence by the corresponding amino acids of a human VH domain.

[39] The compound of any one of items [23] to [35], wherein in (B) said humanization of said sequence is by replacing up to 10 amino acids of said sequence by the corresponding amino acids of a human VH domain. [40] The compound of any one of items [23] to [35], wherein in (B) said humanization of said sequence is by replacing up to 5 amino acids of said sequence by the corresponding amino acids of a human VH domain.

[41] The compound of any one of items [23] to [35], wherein in (B) said humanization of said sequence is by replacing up to 3 amino acids of said sequence by the corresponding amino acids of a human VH domain.

[42] The compound of any one of items [23] to [35], wherein in (B) said humanization of said sequence is by replacing up to 2 amino acids of said sequence by the corresponding amino acids of a human VH domain.

[43] The compound of any one of items [23] to [35], wherein in (B) said humanization of said sequence is by replacing one amino acid of said sequence by the corresponding amino acid of a human VH domain.

[44] The compound of any one of items [23] to [43], wherein in (B) said humanization is within the framework regions of said VHH antibody domain and/or within the CDRs of said VHH antibody domain.

[45] The compound of any one of items [23] to [43], wherein in (B) said humanization is within the framework regions of said VHH antibody domain, but not within the CDRs of said VHH antibody domain.

[46] The compound of any one of items [23] to [43], wherein in (B) said humanization is within the CDRs of said VHH antibody domain, but not within the framework regions of said VHH antibody domain.

[47] The compound of any one of items [23] to [44] or [46], wherein in (B) said humanization within the CDRs of said VHH antibody domain is within CDR1, CDR2 and/or CDR3. [48] The compound of any one of items [23] to [44] or [46] to [47], wherein in (B) said humanization within the CDRs of said VHH antibody domain is within CDR1 and/or CDR2.

[49] The compound of any one of items [23] to [44] or [46] to [48], wherein in (B) said humanization within the CDRs of said VHH antibody domain is within CDR1.

[50] The compound of any one of items [23] to [44] or [46] to [49], wherein in (B) said humanization within the CDRs of said VHH antibody domain is within CDR2.

[51] The compound of any one of items [23] to [50], wherein in (B) said humanization within the CDRs of said VHH antibody domain is not within CDR3.

[52] The compound of any one of items [23] to [51], wherein in (C) the modification is the replacement, addition or deletion of up to 20 amino acids.

[53] The compound of any one of items [23] to [51], wherein in (C) the modification is the replacement, addition or deletion of up to 15 amino acids.

[54] The compound of any one of items [23] to [51], wherein in (C) the modification is the replacement, addition or deletion of up to 10 amino acids.

[55] The compound of any one of items [23] to [51], wherein in (C) the modification is the replacement, addition or deletion of up to 5 amino acids.

[56] The compound of any one of items [23] to [51], wherein in (C) the modification is the replacement, addition or deletion of up to 3 amino acids.

[57] The compound of any one of items [23] to [51], wherein in (C) the modification is the replacement, addition or deletion of up to 2 amino acids.

[58] The compound of any one of items [23] to [51], wherein in (C) the modification is the replacement, addition or deletion of one amino acid. [59] The compound of any one of items [23] to [58], wherein the modification in (C) comprises only the replacement, but not the addition or deletion of amino acids.

[60] The compound of any one of items [23] to [59], wherein in (A) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29, VHH NKp46.31 and VHH NKp46.34 as shown in the Table of CDRs.

[61] The compound of any one of items [23] to [60], wherein in (B) to (D)

(a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH

NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH

NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH

NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH

NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29, VHH

NKp46.31 and VHH NKp46.34 as shown in the Table of CDRs;

(c) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with modification, wherein the modification is the replacement, addition or deletion of up to three amino acids in CDR1, the replacement, addition or deletion of up to three amino acids in CDR2 and/or the replacement, addition or deletion of up to three amino acids in CDR3.

[62] The compound of item [61], wherein the modification in (b) is that the sequence of CDR1 and/or CDR2, but not the sequence of CDR3 is humanized.

[63] The compound of any one of items [61] or [62], wherein the modification in (b) is that the sequence of CDR1 is humanized, but not the sequence of CDR2 and CDR3.

[64] The compound of any one of items [61] or [62], wherein the modification in (b) is that the sequence of CDR2 is humanized, but not the sequence of CDR1 and CDR3.

[65] The compound of any one of items [61] to [64], wherein the modification in (b) is that the sequence of one, but not more than one of CDR1, CDR2 and CDR3 is humanized.

[66] The compound of any one of items [61] to [65], wherein said humanization of said CDR(s) is by replacing at least one amino acid in the sequence of said CDR by the corresponding amino acid of a human VH domain.

[67] The compound of any one of items [61] to [66], wherein said humanization of said CDR(s) is by replacing up to three amino acids in the sequence of said CDR by the corresponding amino acid of a human VH domain.

[68] The compound of any one of items [61] to [66], wherein said humanization of said CDR(s) is by replacing up to three amino acids in the sequence of CDR1 and/or CDR2 and up to one amino acid in the sequence of CDR3 by the corresponding amino acid of a human VH domain.

[69] The compound of any one of items [61] to [66], wherein said humanization of said CDR(s) is by replacing up to two amino acids in the sequence of said CDR by the corresponding amino acid of a human VH domain. [70] The compound of any one of items [61] to [66], wherein said humanization of said CDR(s) is by replacing up to two amino acids in the sequence of CDR1 and/or CDR2 and up to one amino acid in the sequence of CDR3 by the corresponding amino acid of a human VH domain.

[71] The compound of any one of items [61] to [70], wherein said humanization of said CDR(s) is by replacing one amino acid in the sequence of said CDR by the corresponding amino acid of a human VH domain.

[72] The compound of any one of items [61] to [71], wherein the modification in (c) is the replacement, addition or deletion of up to three amino acids in CDR1, the replacement, addition or deletion of up to three amino acids in CDR2 and/or the replacement, addition or deletion of up to two amino acids in CDR3.

[73] The compound of any one of items [61] to [71], wherein the modification in (c) is the replacement, addition or deletion of up to three amino acids in CDR1, the replacement, addition or deletion of up to three amino acids in CDR2 and/or the replacement, addition or deletion of up to one amino acid in CDR3.

[74] The compound of any one of items [61] to [71], wherein the modification in (c) is the replacement, addition or deletion of up to three amino acids in CDR1, and/or the replacement, addition or deletion of up to three amino acids in CDR2, wherein the sequence of CDR3 is unmodified.

[75] The compound of any one of items [61] to [71], wherein the modification in (c) is the replacement, addition or deletion of up to two amino acids in CDR1, the replacement, addition or deletion of up to two amino acids in CDR2 and/or the replacement, addition or deletion of up to two amino acids in CDR3;

[76] The compound of any one of items [61] to [71], wherein the modification in (c) is the replacement, addition or deletion of up to two amino acids in CDR1; the replacement, addition or deletion of up to two amino acids in CDR2; and/or the replacement, addition or deletion of up to one amino acid in CDR3.

[77] The compound of any one of items [61] to [71], wherein the modification in (c) is the replacement, addition or deletion of up to two amino acids in CDR1 and/or the replacement, addition or deletion of up to two amino acids in CDR2; wherein the sequence of CDR3 is unmodified.

[78] The compound of any one of items [61] to [71], wherein the modification in (c) is the replacement, addition or deletion of up to two amino acids in CDR1, wherein the sequence of CDR2 and CDR3 is unmodified.

[79] The compound of any one of items [61] to [71], wherein the modification in (c) is the replacement, addition or deletion of up to two amino acids in CDR2, wherein the sequence of CDR1 and CDR3 is unmodified.

[80] The compound of any one of items [61] to [71], wherein the modification in (c) is the replacement, addition or deletion of up to one amino acid in CDR1; the replacement, addition or deletion of up to one amino acid in CDR2; and/or the replacement, addition or deletion of up to one amino acid in CDR3.

[81] The compound of any one of items [61] to [71], wherein the modification in (c) is the replacement, addition or deletion of one amino acid in CDR1 and/or the replacement, addition or deletion of one amino acid in CDR2; wherein the sequence of CDR3 is unmodified.

[82] The compound of any one of items [61] to [71], wherein the modification in (c) is the replacement, addition or deletion of one amino acid in CDR1, wherein the sequence of CDR2 and CDR3 is unmodified. [83] The compound of any one of items [61] to [71], wherein the modification in (c) is the replacement, addition or deletion of one amino acid in CDR2, wherein the sequence of CDR1 and CDR3 is unmodified.

[84] The compound of any one of items [61] to [83], wherein the modification in (c) comprises only the replacement, but not the addition or deletion of amino acids.

[85] The compound according to any one of items [1] to [84], wherein said compound comprises a VHH antibody domain (not only a fragment of a VHH antibody domain).

[86] The compound according to any one of items [1] to [84], wherein said compound consists of a fragment of a VHH antibody domain (not a full-length VHH antibody domain).

[87] The compound according to any one of items [1] to [84] or [86], wherein said fragment consists of at least 100 amino acids.

[88] The compound according to any one of items [1] to [84] or [86], wherein said fragment consists of at least 105 amino acids.

[89] The compound according to any one of items [1] to [84] or [86], wherein said fragment consists of at least 110 amino acids.

[90] The compound according to any one of items [1] to [84] or [86], wherein said fragment consists of at least 115 amino acids.

[91] The compound according to any one of items [1] to [90], wherein said compound is capable of specifically binding to NKp46.

[92] The compound according to any one of items [1] to [91], wherein said VHH antibody domain or fragment thereof is capable of specifically binding to NKp46.

Ill [93] The compound according to any one of items [1] to [92], wherein said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a KD of IxlO'⁶ M or stronger.

[94] The compound according to any one of items [1] to [92], wherein said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a KD of IxlO'⁷ M or stronger.

[95] The compound according to any one of items [1] to [92], wherein said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a KD of 3.0x10' 8 M or stronger.

[96] The compound according to any one of items [1] to [92], wherein said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a KD of IxlO'⁸ M or stronger.

[97] The compound according to any one of items [1] to [92], wherein said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a KD of IxlO'⁹ M or stronger.

[98] The compound according to any one of items [1] to [97], wherein said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_on of IxlO⁴ 1/Ms or higher.

[99] The compound according to any one of items [1] to [97], wherein said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_on of IxlO⁵ 1/Ms or higher.

[100] The compound according to any one of items [1] to [97], wherein said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_on of 5xl0⁵ 1/Ms or higher. [101] The compound according to any one of items [1] to [100], wherein said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_Off of 5x1 O'³ 1/s or lower.

[102] The compound according to any one of items [1] to [100], wherein said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_Off of IxlO'³ 1/s or lower.

[103] The compound according to any one of items [1] to [100], wherein said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_Off of IxlO'⁴ 1/s or lower.

[104] The compound according to any one of items [1] to [100], wherein said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_Off of IxlO'⁵ 1/s or lower.

[105] The compound according to any one of items [1] to [100], wherein said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_Off of IxlO'⁶ 1/s or lower.

[106] The compound according to any one of items [1] to [100], wherein said VHH antibody domain or fragment thereof binds to recombinant human NKp46 with a k_Off of IxlO'⁷ 1/s or lower.

[107] The compound according to any one of items [1] to [106], wherein in (b) and (c) the VHH antibody domain or fragment thereof binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 5 weaker than the binding of the corresponding VHH antibody domain without modification.

[108] The compound according to any one of items [1] to [106], wherein in (b) and (c) the VHH antibody domain or fragment thereof binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 2 weaker than the binding of the corresponding VHH antibody domain without modification. [109] The compound according to any one of items [1] to [106], wherein in (b) and (c) the VHH antibody domain or fragment thereof binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 1.5 weaker than the binding of the corresponding VHH antibody domain without modification.

[110] The compound according to any one of items [1] to [109], wherein in (b) and (c) the VHH antibody domain or fragment thereof binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 5 stronger than the binding of the corresponding VHH antibody domain without modification.

[111] The compound according to any one of items [1] to [109], wherein in (b) and (c) the VHH antibody domain or fragment thereof binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 2 stronger than the binding of the corresponding VHH antibody domain without modification.

[112] The compound according to any one of items [1] to [109], wherein in (b) and (c) the VHH antibody domain or fragment thereof binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 1.5 stronger than the binding of the corresponding VHH antibody domain without modification.

[113] The compound according to any one of items [23] to [112], wherein in (B) and (C) the VHH antibody domain binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 5 weaker than the binding of the corresponding VHH antibody domain without modification.

[114] The compound according to any one of items [23] to [112], wherein in (B) and (C) the VHH antibody domain binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 2 weaker than the binding of the corresponding VHH antibody domain without modification.

[115] The compound according to any one of items [23] to [112], wherein in (B) and (C) the VHH antibody domain binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 1.5 weaker than the binding of the corresponding VHH antibody domain without modification. [116] The compound according to any one of items [23] to [115], wherein in (B) and (C) the VHH antibody domain binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 5 stronger than the binding of the corresponding VHH antibody domain without modification.

[117] The compound according to any one of items [23] to [115], wherein in (B) and (C) the VHH antibody domain binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 2 stronger than the binding of the corresponding VHH antibody domain without modification.

[118] The compound according to any one of items [23] to [115], wherein in (B) and (C) the VHH antibody domain binds to recombinant human NKp46 with an affinity (KD value) that is by not more than a factor of 1.5 stronger than the binding of the corresponding VHH antibody domain without modification.

[119] The compound according to any one of items [23] to [118], wherein in (D) the affinity (KD value) of the binding of the VHH antibody domain to human NKp46 is by not more than a factor of 5 weaker than the affinity (KD value) of the binding to human NKp46 of a VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has the highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

[120] The compound according to any one of items [23] to [118], wherein in (D) the affinity (KD value) of the binding of the VHH antibody domain to human NKp46 is by not more than a factor of 2 weaker than the affinity (KD value) of the binding to human NKp46 of a VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has the highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

[121] The compound according to any one of items [23] to [118], wherein in (D) the affinity (KD value) of the binding of the VHH antibody domain to human NKp46 is by not more than a factor of 1.5 weaker than the affinity (KD value) of the binding to human NKp46 of a VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has the highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

[122] The compound according to any one of items [23] to [121], wherein in (D) the affinity (KD value) of the binding of the VHH antibody domain to human NKp46 is by not more than a factor of 5 stronger than the affinity (KD value) of the binding to human NKp46 of a VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has the highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

[123] The compound according to any one of items [23] to [121], wherein in (D) the affinity (KD value) of the binding of the VHH antibody domain to human NKp46 is by not more than a factor of 2 stronger than the affinity (KD value) of the binding to human NKp46 of a VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has the highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

[124] The compound according to any one of items [23] to [121], wherein in (D) the affinity (KD value) of the binding of the VHH antibody domain to human NKp46 is by not more than a factor of 1.5 stronger than the affinity (KD value) of the binding to human NKp46 of a VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has the highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

[125] The compound according to any one of items [92] to [124], wherein said KD value/said k_on rate/said k_Off rate is measured by kinetic measurements by biolayer interferometry at 25°C and 1000 rpm in KB Buffer (PBS + 0.1 % Tween-20 + 1% BSA).

[126] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29 or VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 1.0E-08 M or stronger was observed for binding to NKp46.)

[127] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.8, VHHNKp46.9, VHHNKp46.10, VHH NKp46. l 1, VHHNKp46.12, VHHNKp46.13, VHH NKp46.14, VHH NKp46.21, VHH NKp46.23, VHH NKp46.25, VHH NKp46.28, VHH NKp46.29 or VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 5.0E-09 M or stronger was observed for binding to NKp46.)

[128] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.13, VHH NKp46.14, VHH NKp46.21, VHH NKp46.29 or VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 2.0E-09 M or stronger was observed for binding to NKp46.)

[129] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.5, VHH NKp46.10 or VHH NKp46.29 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 1.0E-09 M or stronger was observed for binding to NKp46.)

[130] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.10 or VHH NKp46.29 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 5.0E-10 M or stronger was observed for binding to NKp46.) [131] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.29 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 1.0E-11 M or stronger was observed for binding to NKp46.)

[132] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.11 , VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.25, VHH NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29 or VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 1.0E+05 1/Ms or higher was observed for binding to NKp46.)

[133] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.8, VHH NKp46.9, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.18, VHH NKp46.21, VHH NKp46.22, VHH NKp46.25, VHH NKp46.26, VHH NKp46.27 or VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 2.0E+05 1/Ms or higher was observed for binding to NKp46.)

[134] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.1, VHH NKp46.4, VHH NKp46.5, VHH NKp46.8, VHH NKp46.9, VHH NKp46.12, VHH NKp46.13 or VHH NKp46.21 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 5.0E+05 1/Ms or higher was observed for binding to NKp46.) [135] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.1, VHH NKp46.4, VHH NKp46.5, VHH NKp46.9 or VHH NKp46.21 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 6.0E+05 1/Ms or higher was observed for binding to NKp46.)

[136] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.21 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 7.0E+05 1/Ms or higher was observed for binding to NKp46.)

[137] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHHNKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29, VHH NKp46.31 or VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 5.0E-03 1/Ms or lower was observed for binding to NKp46.)

[138] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.14, VHH NKp46.20, VHH NKp46.23, VHH NKp46.24, VHH NKp46.28, VHH NKp46.29, VHH NKp46.31 or VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 1.0E-03 1/Ms or lower was observed for binding to NKp46.) [139] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.2, VHH NKp46.5, VHH NKp46.6, VHH NKp46.10, VHH NKp46.23, VHH NKp46.28 or VHH NKp46.29 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 5.0E-04 1/Ms or lower was observed for binding to NKp46.)

[140] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.10 or VHH NKp46.29 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 1.0E-07 1/Ms or lower was observed for binding to NKp46.)

[141] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.2, VHH NKp46.4, VHHNKp46.8, VHHNKp46.13, VHHNKp46.15, VHHNKp46.18, VHHNKp46.21 or VHH NKp46.25 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain belong to a specific epitope bin (epitope bin 1) with common characteristics.)

[142] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.22 or VHH NKp46.26 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain are directed to an epitope that overlaps with epitope bin 1.)

[143] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.2, VHH NKp46.4, VHHNKp46.8, VHHNKp46.13, VHHNKp46.15, VHHNKp46.18, VHHNKp46.21, VHH NKp46.22, VHH NKp46.25 or VHH NKp46.26 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain either belong to epitope bin 1 or are directed to an epitope that overlaps with epitope bin 1.) [144] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises the VHH sequence VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain belong to a specific epitope bin (epitope bin 2).)

[145] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.4, VHH NKp46.17, VHH NKp46.23, VHH NKp46.24, VHH NKp46.29 or VHH NKp46.31 shown in the Table of VHH Sequences. (As can be seen from Example 3, compounds with such a VHH antibody domain allowed to prepare compounds that showed strong killing activity in the assay of Fig. 2a.)

[146] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.2, VHH NKp46.4, VHHNKp46.8, VHHNKp46.13, VHHNKp46.15, VHHNKp46.18, VHHNKp46.21, VHH NKp46.22, VHH NKp46.25, VHH NKp46.26 or VHH NKp46.34 shown in the Table of VHH Sequences. (As can be seen from Example 3, compounds with such a VHH antibody domain allowed to prepare compounds that showed strong killing activity in the assay of Fig. 2B and 3 A.)

[147] The compound of any one of items [23] to [125], wherein said VHH antibody domain of (A) comprises any one of the VHH sequences VHH NKp46.2, VHH NKp46.18 or VHH NKp46.21 shown in the Table of VHH Sequences. (As can be seen from Example 3, Fig. 2B and 3A, compounds with such a VHH antibody domain allowed to prepare compounds that showed particularly strong killing activity.)

[148] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29 and VHH NKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 1.0E-08 M or stronger was observed for binding to NKp46.)

[149] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.21, VHH NKp46.23, VHH NKp46.25, VHH NKp46.28, VHH NKp46.29 and VHH NKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 5.0E-09 M or stronger was observed for binding to NKp46.)

[150] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.13, VHH NKp46.14, VHH NKp46.21, VHH NKp46.29 and VHHNKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 2.0E-09 M or stronger was observed for binding to NKp46.)

[151] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.5, VHH NKp46.10 and VHH NKp46.29 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 1.0E-09 M or stronger was observed for binding to NKp46.) [152] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.10 and VHH NKp46.29 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 5.0E-10 M or stronger was observed for binding to NKp46.)

[153] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.29 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a KD in the range of 1.OE-11 M or stronger was observed for binding to NKp46.)

[154] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.25, VHH NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29 and VHH NKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 1.0E+05 1/Ms or higher was observed for binding to NKp46.)

[155] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.8, VHH NKp46.9, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.18, VHH NKp46.21, VHH NKp46.22, VHH NKp46.25, VHH NKp46.26, VHH NKp46.27 and VHH NKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 2.0E+05 1/Ms or higher was observed for binding to NKp46.)

[156] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.4, VHH NKp46.5, VHH NKp46.8, VHH NKp46.9, VHH NKp46.12, VHH NKp46.13 and VHH NKp46.21 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 5.0E+05 1/Ms or higher was observed for binding to NKp46.)

[157] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.4, VHH NKp46.5, VHH NKp46.9 and VHH NKp46.21 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 6.0E+05 1/Ms or higher was observed for binding to NKp46.)

[158] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.21 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_on in the range of 7.0E+05 1/Ms or higher was observed for binding to NKp46.)

[159] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29, VHH NKp46.31 and VHH NKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 5.0E-03 1/Ms or lower was observed for binding to NKp46.)

[160] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.8, VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.14, VHH NKp46.20, VHH NKp46.23, VHH NKp46.24, VHH NKp46.28, VHH NKp46.29, VHH NKp46.31 and VHH NKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 1.0E-03 1/Ms or lower was observed for binding to NKp46.)

[161] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHHNKp46.2, VHH NKp46.5, VHHNKp46.6, VHHNKp46.10, VHH NKp46.23, VHH NKp46.28 and VHHNKp46.29 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 5.0E-04 1/Ms or lower was observed for binding to NKp46.)

[162] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.10 and VHH NKp46.29 as shown in the Table of CDRs. (As can be seen e.g. from Example 2, Table I, for compounds with such a VHH antibody domain a k_Off in the range of 1.0E-07 1/Ms or lower was observed for binding to NKp46.)

[163] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHHNKp46.2, VHH NKp46.4, VHHNKp46.8, VHHNKp46.13, VHH NKp46.15, VHH NKp46.18, VHH NKp46.21 and VHH NKp46.25 as shown in the Table of CDRs. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain belong to a specific epitope bin (epitope bin 1) with common characteristics.)

[164] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.22 and VHH NKp46.26 as shown in the Table of CDRs. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain are directed to an epitope that overlaps with epitope bin 1.)

[165] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHHNKp46.2, VHH NKp46.4, VHHNKp46.8, VHHNKp46.13, VHH NKp46.15, VHH NKp46.18, VHHNKp46.21, VHH NKp46.22, VHH NKp46.25 and VHH NKp46.26 as shown in the Table of CDRs. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain either belong to epitope bin 1 or are directed to an epitope that overlaps with epitope bin 1.)

[166] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH NKp46.34 as shown in the Table of CDRs. (As can be seen e.g. from Example 4, compounds with such a VHH antibody domain belong to a specific epitope bin (epitope bin 2).) [167] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.4, VHH NKp46.17, VHH NKp46.23, VHH NKp46.24, VHH NKp46.29 and VHH NKp46.31 as shown in the Table of VHH Sequences. (As can be seen from Example 3, compounds with such a VHH antibody domain allowed to prepare compounds that showed strong killing activity in the assay of Fig. 2a.)

[168] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHHNKp46.2, VHH NKp46.4, VHHNKp46.8, VHH NKp46.13, VHH NKp46.15, VHH NKp46.18, VHH NKp46.21, VHH NKp46.22, VHH NKp46.25, VHH NKp46.26 and VHH NKp46.34 as shown in the Table of VHH Sequences. (As can be seen from Example 3, compounds with such a VHH antibody domain allowed to prepare compounds that showed strong killing activity in the assay of Fig. 2B and 3A.)

[169] The compound of any one of items [1] to [22] or [61] to [147], wherein in (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.2, VHH NKp46.18 and VHH NKp46.21 as shown in the Table of VHH Sequences. (As can be seen from Example 3, Fig. 2B and 3A, compounds with such a VHH antibody domain allowed to prepare compounds that showed particularly strong killing activity.)

[170] The compound according to any one of items [1] to [169], wherein said VHH antibody domain or fragment thereof competes with VHH NKp46.2 for binding to human NKp46.

[171] The compound according to any one of items [1] to [169], wherein said VHH antibody domain or fragment thereof partially competes with VHH NKp46.2 for binding to human NKp46. [172] The compound according to any one of items [1] to [169], wherein said VHH antibody domain or fragment thereof does not compete with VHH NKp46.2 for binding to human NKp46.

[173] The compound according to any one of items [1] to [172], wherein said VHH antibody domain or fragment thereof competes with VHH NKp46.34 for binding to human NKp46.

[174] The compound according to any one of items [1] to [162], wherein said VHH antibody domain or fragment thereof partially competes with VHH NKp46.34 for binding to human NKp46.

[175] The compound according to any one of items [1] to [172], wherein said VHH antibody domain or fragment thereof does not compete with VHH NKp46.34 for binding to human NKp46.

[176] The compound according to any one of items [1] to [175], wherein said humanization is by germlining.

[177] The compound according to item [176], wherein said germlining involves (preferably consists of)

[178] The compound according to any one of items [1] to [177], wherein said compound with humanization binds to human NKp46 with an affinity (KD value) that is not weaker by a factor of more than 5 compared to the binding of a corresponding compound without humanization to human NKp46.

[179] The compound according to any one of items [1] to [173], wherein said compound with humanization binds to human NKp46 with an affinity (KD value) that is not weaker by a factor of more than 2 compared to the binding of a corresponding compound without humanization to human NKp46.

[180] The compound according to any one of items [1] to [177], wherein said compound with humanization binds to human NKp46 with an affinity (KD value) that is not weaker than the binding of a corresponding compound without humanization to human NKp46.

[181] The compound according to any one of items [1] to [177], wherein said affinity is determined by KD measurement.

[182] The compound according to any one of items [1] to [181], wherein said compound is a molecule.

[183] The compound according to any one of items [1] to [182], wherein said compound comprises or is a protein.

[184] The compound according to any one of items [1] to [183], wherein said compound is a protein.

[185] The compound according to any one of items [23] to [183], wherein said compound comprises a VHH antibody domain or fragment thereof according to (A), (B) or (C).

[186] The compound according to any one of items [23] to [183], wherein said compound comprises a VHH antibody domain or fragment thereof according to (A), (B) or (D).

[187] The compound according to any one of items [23] to [183], wherein said compound comprises a VHH antibody domain or fragment thereof according to (A), (C) or (D).

[188] The compound according to any one of items [23] to [183], wherein said compound comprises a VHH antibody domain or fragment thereof according to (A) or (B).

[189] The compound according to any one of items [23] to [183], wherein said compound comprises a VHH antibody domain or fragment thereof according to (A) or (C). [190] The compound according to any one of items [23] to [183], wherein said compound comprises a VHH antibody domain or fragment thereof according to (A) or (D).

[191] The compound according to any one of items [23] to [183], wherein said compound comprises a VHH antibody domain or fragment thereof according to (A).

[192] The compound according to any one of items [23] to [183], wherein said compound comprises a VHH antibody domain or fragment thereof according to (B).

[193] The compound according to any one of items [1] to [22] or [61] to [192], wherein said VHH antibody domain or fragment thereof comprises complementarity determining regions according to (a) or (b).

[194] The compound according to any one of items [1] to [22] or [61] to [192], wherein said VHH antibody domain or fragment thereof comprises complementarity determining regions according to (a) or (c).

[195] The compound according to any one of items [1] to [22] or [61] to [192], wherein said VHH antibody domain or fragment thereof comprises complementarity determining regions according to (a).

[196] The compound according to any one of items [1] to [22] or [61] to [192], wherein said VHH antibody domain or fragment thereof comprises complementarity determining regions according to (b).

[197] The compound according to any one of items [1] to [173], wherein said compound further comprises a targeting moiety.

[198] The compound according to any one of items [1] to [174], wherein all components of said compound are covalently linked. [199] The compound according to any one of items [174] or [175], wherein said targeting moiety is a molecular group that specifically binds to a target molecule or fragment thereof.

[200] The compound according to item [199], wherein said target molecule is a receptor at the surface of a cell.

[201] The compound according to any one of items [199] or [200], wherein said target molecule is an antigen that is present on the surface of a target cell.

[202] The compound according to any one of items [197] to [201], wherein said targeting moiety is a protein, a peptide, a peptide mimetic, a nucleic acid, an oligonucleotide or a small molecule.

[203] The compound according to any one of items [197] to [202], wherein said targeting moiety comprises or is a protein.

[204] The compound according to any one of items [197] to [203], wherein said targeting moiety comprises a protein.

[205] The compound according to any one of items [197] to [204], wherein said targeting moiety is a protein.

[206] The compound according to any one of items [197] to [205], wherein said targeting moiety is a protein ligand that specifically binds to a receptor at the surface of a cell.

[207] The compound according to any one of items [197] to [205], wherein said targeting moiety is an antibody or an antigen-binding fragment thereof.

[208] The compound according to any one of items [197] to [207], wherein said targeting moiety is capable of specifically binding to a tumor-associated antigen.

[209] The compound according to any one of items [1] to [208], wherein said compound is a bispecific antibody. [210] The compound according to any one of items [1] to [186], wherein said compound is a bispecific antibody prepared by the SEED (strand-exchange engineered domain) technology.

[211] The compound according to any one of items [209] or [210], wherein one binding site of said bispecific antibody is formed by said VHH antibody domain or fragment thereof and one binding site of said bispecific antibody is formed by said targeting moiety.

[212] The compound according to any one of items [207] to [211], wherein said antigenbinding fragment is selected from the group consisting of a Fab, a Fab', a (Fab')2, a Fv, a scFv, a diabody and a VHH.

[213] The compound according to any one of items [207] to [211], wherein said antigenbinding fragment is selected from the group consisting of a Fab, a Fab', a (Fab')2 and a Fv.

[214] The compound according to any one of items [207] to [211], wherein said antigenbinding fragment is a Fab.

[215] The compound according to any one of items [207] to [211], wherein said antigenbinding fragment is selected from the group consisting of a scFv, a diabody and a VHH.

[216] The compound according to any one of items [207] to [215], wherein said antigenbinding fragment is an antigen-binding fragment of an antibody with the SEED (strand-exchange engineered domain) format.

[217] The compound according to any one of items [197] to [216], wherein said targeting moiety is capable of specifically binding to an antigen that is present on the surface of a target cell. [218] The compound according to any one of items [207] to [217], wherein said antibody is an antibody against an antigen that is present on the surface of a target cell.

[219] The compound according to any one of items [207] to [218], wherein said antigenbinding fragment is an antigen-binding fragment of an antibody against an antigen that is present on the surface of a target cell.

[220] The compound according to any one of items [217] to [219], wherein said binding of said targeting moiety to said antigen that is present on the surface of said target cell allows to recruit the compound specifically to said target cell.

[221] The compound according to any one of items [201] to [220], wherein said antigen that is present on the surface of said target cell is a tumor-associated antigen.

[222] The compound according to any one of items [197] to [221], wherein said targeting moiety is capable of specifically binding to a tumor-associated antigen.

[223] The compound according to any one of items [208] to [222], wherein said tumor- associated antigen is an antigen that is present on the surface of a tumor cell.

[224] The compound according to any one of items [208] to [222], wherein said tumor- associated antigen is EGFR (epidermal growth factor receptor).

[225] The compound according to any one of items [1] to [224], wherein said compound is a bispecific or multispecific molecule.

[226] The compound according to any one of items [1] to [225], wherein said compound comprises an antibody Fc region.

[227] The compound according to any one of items [1] to [225], wherein said compound comprises an antibody Fc region competent in Fc receptor binding.

[228] The compound according to any one of items [1] to [227], wherein said compound comprises an antibody Fc region that is not competent in Fc receptor binding. [229] The compound according to any one of items [1] to [228], wherein said compound does not comprise an effector-competent antibody Fc region.

[230] The compound according to any one of items [1] to [228], wherein said compound comprises an effector-competent antibody Fc region.

[231] The compound according to any one of items [1] to [230], wherein said compound does not comprise an antibody Fc region capable of inducing ADCC (antibodydependent cellular cytotoxicity).

[232] The compound according to any one of items [1] to [230], wherein said compound comprises an antibody Fc region capable of inducing ADCC.

[233] The compound according to any one of items [1] to [232], wherein said compound comprises an antibody Fc region that is not capable of inducing ADCC.

[234] The compound according to any one of items [1] to [233], wherein said compound is capable of inducing both FcyRIIIa signalling and positive (i.e. NK cell activating) NKp46 signalling.

[235] The compound according to any one of items [1] to [234], wherein said compound is capable of specifically binding to NKp46 on NK cells.

[236] The compound according to any one of items [1] to [235], wherein said compound is capable of activating NK cells by binding to NKp46 on said NK cells.

[237] The compound according to any one of items [243] to [236], wherein binding of said compound to NKp46 on NK cells activates said NK cells.

[238] The compound according to any one of items [1] to [237], wherein said compound is an agonist of NKp46. [239] The compound according to any one of items [1] to [238], wherein said compound is, upon binding of said VHH antibody domain or fragment thereof to NKp46 on NK cells, capable of activating NK cells.

[240] The compound according to any one of items [1] to [239], wherein binding of said compound to NKp46 on NK cells activates said NK cells.

[241 ] The compound according to any one of items [211 ] to [240], wherein activation of NK cells is measured by measuring expression of the activation marker CD69 by flow cytometry.

[242] The compound according to any one of items [1] to [240], wherein said compound comprises one NKp46-specific VHH domain and one targeting moiety, wherein said targeting moiety is a Fab.

[243] The compound according to any one of items [1] to [240], wherein said compound comprises two NKp46-specific VHH domains and one targeting moiety, wherein said targeting moiety is a Fab.

[244] The compound according to any one of items [1] to [240], wherein said compound comprises two NKp46-specific VHH domains and two targeting moieties, wherein said targeting moieties are Fabs.

[245] The compound according to any one of items [1] to [240], wherein said compound comprises one NKp46-specific VHH domain and two targeting moieties, wherein said targeting moieties are Fabs.

[246] The compound according to any one of items [242] to [245], wherein said Fab is/said Fabs are located at the N-terminus of the molecule.

[247] The compound according to any one of items [242] to [245], wherein said NKp46- specific VHH domain is/said NKp46-specific VHH domains are located at the N- terminus of the compound. [248] The compound according to any one of items [242] to [245], wherein said NKp46- specific VHH domain is/said NKp46-specific VHH domains are located at the C- terminus of the compound.

[249] The compound according to any one of items [1] to [248], wherein said compound shows cytotoxic activity in a ⁵¹Cr release assay.

[250] The compound according to item [249], wherein said ⁵¹Cr release assay is carried out as a 4 h ⁵¹Cr release assay, wherein human PBMCs are used as effector cells at effector- to-target cell (E:T) ratios of 80: 1, and wherein a higher percent lysis indicates improved cytotoxic activity.

[251] The compound according to any one of items [249] to [250], wherein said ⁵¹Cr release assay is carried out as described in Repp et al., 2011.

[252] The compound according to any one of items [1] to [251], wherein binding of said compound to NKp46 on NK cells results in the release of interferon-y (IFN-y).

[253] The compound according to item [252], wherein said release of IFN-y is measured as follows: isolated human NK cells are incubated overnight in medium containing 100 U/ml recombinant human interleukin-2, in different wells A431 cells are seeded and incubated for 3 h, the compound is added to a final concentration of 85 nM followed by addition of NK cells at an E:T ratio of 5: 1, human IFN-y is analyzed in the culture supernatant after 24 h by ELISA.

[254] The compound according to any one of items [1] to [253], wherein binding of said compound to NKp46 on NK cells results in the release of tumor necrosis factor-a (TNF-a).

[255] The compound according to item [254], wherein said release of TNF-a is measured as follows: isolated human NK cells are incubated overnight in medium containing 100 U/ml recombinant human interleukin-2, in different wells A431 cells are seeded and incubated for 3 h, the compound is added to a final concentration of 85 nM followed by addition of NK cells at an E:T ratio of 5: 1, human TNF-a is analyzed in the culture supernatant after 24 h by ELISA.

[256] A pharmaceutical composition comprising the compound according to any one of items [1] to [255],

[257] The pharmaceutical composition according to item [256], wherein said pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent and/or excipient.

[258] The pharmaceutical composition according to any one of items [256] to [257], wherein said pharmaceutical composition further includes at least one additional adjuvant, antioxidant, buffering agent, bulking agent, colorant, emulsifier, filler, flavoring agent, preservative, stabilizer, suspending agent and/or other customary pharmaceutical auxiliary.

[259] A compound according to any one of items [1] to [255] or the pharmaceutical composition according to any one of items [256] to [258] for use as a medicament.

[260] A compound according to any one of items [1] to [255] or the pharmaceutical composition according to any one of items [256] to [258] for use in the treatment of cancer.

[261] A compound according to any one of items [1] to [255] or the pharmaceutical composition according to any one of items [256] to [258] for use in the treatment of a malignant tumor.

[262] A compound according to any one of items [1] to [255] or the pharmaceutical composition according to any one of items [256] to [258], wherein said compound/said pharmaceutical composition is for use in the treatment of a human.

[263] A method for treating a disease in a patient in need thereof, comprising the step of administering to said patient a therapeutically effective amount of the compound according to any one of items [1] to [255] or the pharmaceutical composition according to any one of items [256] to [258], [264] The method according to item [263], wherein said disease is cancer.

[265] The method according to item [263], wherein said disease is a malignant tumor.

[266] The method according to any one of items [263] to [265], wherein said patient is a human.

[267] Use of the compound according to any one of items [1] to [255] or the pharmaceutical composition according to any one of items [256] to [258] for the manufacture of a medicament.

[268] Use of the compound according to any one of items [1] to [255] or the pharmaceutical composition according to any one of items [256] to [258] for the manufacture of a medicament for the treatment of cancer.

[269] Use of the compound according to any one of items [1] to [255] or the pharmaceutical composition according to any one of items [226] to [228] for the manufacture of a medicament for the treatment of a malignant tumor.

[270] The use according to any one of items [267] to [269], wherein said medicament is prepared for administration to a human.

[271] The compound or the pharmaceutical composition for use according to any one of items [260] or [261] or the method according to any one of items [264] or [265] or the use according to any one of items [268] or [269], wherein said cancer or malignant tumor is a human disease.

[272] The compound of any one of items [1] to [255] or [271] or the pharmaceutical composition for use according to any one of items [256] to [258] or [271] or the compound or pharmaceutical composition for use according to any one of items [259] to [262] or [271] or the method according to any one of items [263] to [266] or [271] or the use according to any one of items [267] to [271], wherein said compound binds to NKp46 with a KD of 5* 10'⁶ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA).

[273] The compound of any one of items [1] to [255] or [271] or the pharmaceutical composition for use according to any one of items [256] to [258] or [271] or the compound or pharmaceutical composition for use according to any one of items [259] to [262] or [271] or the method according to any one of items [263] to [266] or [271] or the use according to any one of items [267] to [271], wherein said compound binds to NKp46 with a KD of l >< 10'⁶ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) of NKp46 in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA).

[274] The compound of any one of items [1] to [255] or [271] or the pharmaceutical composition for use according to any one of items [256] to [258] or [271] or the compound or pharmaceutical composition for use according to any one of items [259] to [262] or [271] or the method according to any one of items [263] to [266] or [271] or the use according to any one of items [267] to [271], wherein said compound binds to NKp46 with a KD of 5* 10'⁷ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA).

[275] The compound of any one of items [1] to [255] or [271] or the pharmaceutical composition for use according to any one of items [256] to [258] or [271] or the compound or pharmaceutical composition for use according to any one of items [259] to [262] or [271] or the method according to any one of items [263] to [266] or [271] or the use according to any one of items [267] to [271], wherein said compound binds to NKp46 with a KD of P I O'⁷ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA).

[276] The compound of any one of items [1] to [255] or [271] or the pharmaceutical composition for use according to any one of items [256] to [258] or [271] or the compound or pharmaceutical composition for use according to any one of items [259] to [262] or [271] or the method according to any one of items [263] to [266] or [271] or the use according to any one of items [267] to [271], wherein said compound binds to NKp46 with a KD of 5* 10'⁸ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA).

[277] The compound of any one of items [1] to [255] or [271] or the pharmaceutical composition for use according to any one of items [256] to [258] or [271] or the compound or pharmaceutical composition for use according to any one of items [259] to [262] or [271] or the method according to any one of items [263] to [266] or [271] or the use according to any one of items [267] to [271], wherein said compound binds to NKp46 with a KD of | / I O'^X M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA).

[278] The compound of any one of items [1] to [255] or [271] or the pharmaceutical composition for use according to any one of items [256] to [258] or [271] or the compound or pharmaceutical composition for use according to any one of items [259] to [262] or [271] or the method according to any one of items [263] to [266] or [271] or the use according to any one of items [267] to [271], wherein said compound binds to NKp46 with a KD of 5* 10'⁹ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA).

[279] The compound of any one of items [1] to [255] or [271] or the pharmaceutical composition for use according to any one of items [256] to [258] or [271] or the compound or pharmaceutical composition for use according to any one of items [259] to [262] or [271] or the method according to any one of items [263] to [266] or [271] or the use according to any one of items [267] to [271], wherein said compound binds to NKp46 with a KD of P I O'⁹ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA). EXAMPLES

The following examples describe the preparation and characterization of VHH-based NKp46 binders as disclosed in the present disclosure, as well as related compounds and methods, along with comparative disclosure. It is understood that various embodiments of the disclosure reflected in the examples may be practiced, given the general description provided above. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention.

Example 1

Camelid immunization

For the immunization, two Alpacas (Vicugna pacos) and one Llama (Lama glama) were immunized with recombinant human (rh) NKp46 extracellular domain (ECD; Aero Biosystems; SEQ ID NO: 165; uniprot 076036, NCTR1 HUMAN) at preclinics GmbH, Germany. The immunization protocol comprised four administrations of 300 pg rh NKp46 ECD, each conducted as subcutaneous injections at three sites, over a period of 42 days in total (at dO, dl4, d28 and d35). For this, the antigen was diluted to a stock concentration of 1 mg/ml in PBS and emulsified either with Complete Freund’s Adjuvant, for initial immunization, or with Incomplete Freund’s Adjuvant for subsequent immunizations. Seven days after the final administration (d42), a volume of 100 ml blood per specimen was collected prior to RNA extraction and subsequent cDNA synthesis. All procedures and animal care were in accordance with local animal welfare protection laws and regulation. All procedures involving animals were conducted at preclinics GmbH. Animals remained alive after immunization and final blood collection.

Yeast strains and media

Saccharomyces cerevisiae strain EBY100 (MATa URA3-52 trpl leu2Al his3A200 pep4::HIS3 prblA1.6R canl GAL (pIU211:URA3y) (Thermo Fisher Scientific) was employed for yeast surface display. Cells were cultivated in Yeast Extract-Peptone-Dextrose (YPD) medium composed of 20 g/1 peptone, 20 g/1 dextrose and 10 g/l yeast extract supplemented with 10 mg/ml penicillin-streptomycin (Gibco). After homologous recombination-based cloning, cells harboring library plasmids (pDisp) were cultivated in medium using minimal synthetic defined (SD)-base (Takara Bio) and corresponding dropout mix (Takara Bio) composed of all essential amino acids except for tryptophan (-Trp) for selection, supplemented with 5.4 g/1 Na2HPO4 and 8.6 g/1 NaJfcPC ^x H2O. To induce antibody gene expression, cells were transferred into galactose containing SG dropout medium (-Trp), consisting of SG-base medium (Takara Bio) supplemented with 10% (w/v) polyethylene glycol 8000 (PEG 8000).

Plasmids for yeast surface display and library generation

Gap repair cloning based on homologous recombination in yeast was exploited for the generation of VHH libraries. Protocols for PCR amplification of VHH fragments as well as library construction were carried out as described in Roth et al., 2020. In brief, digestion of the display plasmid pDisp with specific restriction enzyme Bsal followed by genetic fusion of VHH library candidates in frame to Aga2p by replacement of a stuffer sequence due to gap repair cloning allowed for the presentation of sdAb variants on the yeast cell surface. The additional insertion of a HA epitope linked C-terminally to Aga2p on the pDisp backbone enabled the detection of proper full-length VHH presentation on the yeast surface.

Library sorting

For sorting purposes, EBY100 cells were grown overnight in SD medium with dropout mix lacking tryptophan (-Trp) at 30 °C and 120 rpm prior to induction of surface expression by cell transfer into SG medium with dropout mix (-Trp) at 10⁷ cells/ml and 48 h incubation at 20 °C. Antigen binding was detected by indirect immunofluorescence using 1 pM rh his-tagged NKp46 ECD (Aero Biosystems) in combination with anti-his mouse monoclonal detection antibody (SureLight® Allophycocyanin, Abeam, diluted 1 :20). Simultaneous monitoring of full-length VHH surface expression by application of a FITC-labeled rabbit polyclonal anti -HA antibody (Abeam, diluted 1 :20) allowed for a two-dimensional sorting strategy (Fig. IB). The fluorescence activated cell sorting (FACS) procedure was performed on a BD FACSAria™ Fusion cell sorter (BD Biosciences) device. Control samples, i.e., cells incubated with secondary labeling reagents only or cells incubated with secondary labeling reagents and his- tagged NKp46 or unrelated antigen as well as untreated cells were employed in every experiment, allowing for gate adjustment of the desired cell population. Protein expression and purification

After sequencing of FACS enriched populations and subsequent clone selection, the VHH variants were fused A-terminally to the hinge region of Fc immune effector-silenced (eff-) SEED AG chains prior to cloning into pTT5mammalian expression vector (Durocher et al., 2002), ultimately enabling the production of eff- bispecific SEEDbodies (SEEDbody eff-) in combination with eff- humanized Cetuximab Fab on the SEED GA chain for the initial protein characterization. For a more detailed characterization, specific VHHs were also expressed as effector competent SEEDbodies (SEEDbody eff+) and in different orientations and valencies (as eff- versions). For protein expression, Expi293 cells were transiently transfected with respective pTT5 vectors according to the manufacturer’s instructions (Thermo Fisher Scientific). The protein containing supernatants were harvested six days post transfection by centrifugation and purified via MabSelect antibody purification chromatography resin (GE Healthcare), followed by a buffer exchange step to PBS pH 6.8 overnight using Pur-A-Lyzer™ Maxi 3500 Dialysis Kit (Sigma Aldrich). Resulting molecule concentrations were measured using Nanodrop ND- 1000 (Peqlab) after sterile filtration with Ultrafree®-CL GV 0.22 pm centrifugal devices (Merck Millipore).

Protein analytics

For the assessment of protein sample quality regarding target monomer peaks [%], analytical size exclusion chromatography (SEC) was conducted, using 7.5 pg protein per sample on a TSKgel UP-SW3000 column (2 pm, 4.6 x 300 mm, Tosoh Bioscience) in an Agilent HPLC 1260 Infinity system with a flow rate of 0.35 ml/min using 50 mM sodium phosphate, 0.4 M NaCICU pH 6.3 as mobile phase. Hydrophobicity of the different molecules was determined by hydrophobic interaction chromatography (HIC) using 20 pg protein per sample on a TSKgel Butyl-NPR column (2.5 pm, 4.6 x 100 mm, Tosoh Bioscience) in an Agilent HPLC 1260 Infinity system with a flow rate of 0.5 ml/min. Samples were premixed with 50% (v/v) 2 M ammonium sulfate solution prior to injection. A gradient running from mobile phase A (1.2 M ammonium sulfate in PBS) to mobile phase B (50% methanol in O. lx PBS) over 15.0 min at 25°C was applied. Signals were recorded at 214 nm. Anti-PD-Ll Avelumab and anti-EGFR Cetuximab were used as reference molecules. Thermal unfolding of the antibodies was assessed by differential scanning fluorimetry (DSF) on a Prometheus NT.PLEX nanoDSF instrument. Samples were measured in duplicates using nanoDSF grade standard capillaries. A temperature gradient from 20°C to 95°C at a slope of l°C/min was used while recording fluorescence at 350 and 330 nm. Unfolding transition midpoints (Tm) were determined from the first derivative of the fluorescence ratio 350 nm/330 nm.

Molecular Modeling and in silico property prediction

To create homology models of the full length IgGs and VHHs the antibody modeler tool in the molecular modeling software package moe (Mol Operating Enrion 2020.09: Chemical Computing Group Inc.; 2020) was utilized. The generation of IgG-VHH constructs were built by adding linkers via moe’s protein builder, followed by a conformational search of the linker via moe’s linker modeler. Finally, an energy minimization was performed, treating the linker as flexible and the IgG and VHH domains as rigid bodies. Visualization of 3D structures was done with PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrodinger, LLC.).

The in silico developability profile was computed using an internal pipeline termed “Sequence Assessment Using Multiple Optimization Parameters (SUMO)” (Evers et al., 2022). Briefly, this approach automatically generates VHH models based on the provided sequences, identifies the human-likeness by sequence comparison to the most similar human germline sequence, determines structure-based surface-exposed chemical liability motifs (unpaired cysteines, methionines, asparagine deamidation motifs and aspartate deamidation sites) as well as sites susceptible to post-translational modification (N-linked glycosylation). Moreover, a small set of orthogonal computed physico-chemical descriptors including the isoelectric point (pl) of the variable domain, Schrodingers AggScore as predictor for hydrophobicity and aggregation tendency calculated for the complete variable domain as well as the complementaritydetermining regions (CDRs) only and the calculated positive patch energy of the CDRs were determined (Sankar et al., 2018). These scores were complemented with a green to yellow to red color coding, indicating scores within one standard deviation from the mean over a benchmarking dataset of multiple biotherapeutics approved for human application as green, scores above one standard deviation as yellow and those above two standard deviations as red (Ahmed et al., 2021) (Fig. 14). For the AggScore values, these cutoffs were slightly adjusted based on correlation analyses to internal experimental HIC data.

Biolayer interferometry (BLI)

For binding assays with recombinant proteins, the Octet RED96 system (ForteBio, Pall Life Science) was employed using 25 °C and 1000 rpm agitation settings. In order to determine binding kinetics, bispecific molecules were loaded on anti-human Fc (AHC) Biosensors at 3 pg/ml in PBS for 3 min followed by 60 s sensor rinsing in kinetics buffer (KB; PBS + 0.1 % Tween-20 and 1 % bovine serum albumin, BSA). Afterwards, binding to human NKp46 ECD (Aero Biosystems) in decreasing concentrations ranging from 100 nM to 1.56 nM in KB was measured for 300 s followed by dissociation for 300 s in KB. In each experiment, one negative control using irrelevant antigen and a second reference by incubating the antibody in KB instead of the antigen was measured.

Simultaneous binding capacities of NKCEs were measured by loading his-tagged EGFR ECD (produced in-house) on anti-his tips (HIS1K) for 3 min at 3 pg/ml in PBS. After sensor rinsing a first binding step was performed using the respective NKCE at 100 nM, followed by a consecutive association step with 100 nM of NKp46 Fc-fusion protein (Aero Biosystems). Parallel control measurements for each association step of Biosensors incubated in KB instead were utilized.

To analyze competitive binding of VHHs, his-tagged NKp46 ECD was loaded at 3 pg/ml in PBS for 3 min to HIS IK Biosensors followed by 60 s sensor rinsing in KB. Association of the bispecific antibodies (100 nM) for 200 s in KB was followed by an additional association step with a different SEEDbody for another 150 s in KB in presence of 100 nM first analyte. Control values using an unrelated antibody or the same bispecific SEEDbody for both association steps as well as controls using KB buffer were included.

Data was fitted and analyzed with ForteBio data analysis software 8.0 using a 1 : 1 binding model after Savitzky-Golay filtering.

Tumor cell killing assays

A detailed protocol has previously been described by our group and can be found elsewhere (Pekar et al., 2020). In brief, peripheral blood mononuclear cells (PBMCs) were freshly isolated from healthy donors. Subsequently, NK cells were enriched using EasySep™ Human NK Cell Isolation Kit (Stemcell Technologies). Purified NK cells were rested overnight in complete medium supplemented with low dose rh IL-2 (100 U/ml, R&D systems) prior to cell adjustment to 0.625 x 10⁶ viable cells per ml the next day. EGFR overexpressing A431 cells or EGFR negative ExpiCHO™ cells were prepared by cell staining with CellTracker™ Deep Red Dye (ThermoFisher) and seeded into a 384-well clear bottom microtiter plate (Greiner Bio-One) at 2500 cells/well. After a 3 h adherence period, NK effector cells were dispensed to target cells at an effector to target cell (E:T) ratio of 5: 1 before addition of bsAbs at concentrations as indicated followed by incubation for 24 h in the Incucyte® system. As negative control, a monovalent EGFR targeting Fc effector-silenced antibody derivative was used (oa_hu225 SEEDbody eff-). Dead cells were detected by dispensing 0.03 pM SYTOX™ Green Dead Cell Stain (Invitrogen) to the assay. Cell lysis was normalized to maximum lysis induced by Cetuximab or to target cells cultivated with 30 pM staurosporine (Merck Millipore). Overlay signals allowed for analysis of dead target cells only, while subtraction of overlay signals from overall green signal enabled specific analysis of dead NK cells.

NK cell activation assay

To determine specific NK cell activation by herein engineered bsAbs, 20.000 A431 cells/well were seeded in 96-well V-bottom microtiter plates (Thermo Fisher Scientific) and rested 3 h for adherence prior to the addition of 100.000 NK cells/well, resulting in and E:T ratio of 5: 1. Antibodies were added at a final concentration of 50 nM followed by 24 h incubation at 37 °C. For NK surface receptor staining, cells were washed once with PBS + 1 % BSA, followed by incubation with anti-CD69 PE (R&D Systems) and anti-CD56 PE-Cy7 (Beckman Coulter) or respective isotype controls for 1 h on ice. After another washing step, cells were analyzed by flow cytometry employing the IntelliCyt® iQue® Screener Plus system (Sartorius). For measurement and compensation of fluorochromes the IntelliCyt® ForeCyt® Enterprise Client Edition 8.0 (R3) Version 8.0.7430 software (Sartorius) was used. The applied gating strategy is shown in Figure 8.

Data processing and statistical analysis

Graphical and statistical analyses were conducted with GraphPad Prism 8 software. /^J- values were calculated utilizing repeated measures ANOVA and the Bonferroni or Tukey post-test as recommended, or the student’s /-test when appropriate, p < 0.05 were regarded as statistically significant.

All methods in Examples 2 to 6 were carried out as described in Example 1. Example 2

Isolation of NKp46-specific VHH single domain antibodies (sdAbs) / Biochemical and biophysical properties

In order to generate bispecific antibodies (bsAbs) triggering NK cells via NCR NKp46, singledomain antibodies (sdAbs) directed against NKp46 were isolated from camelids. To this end, two Alpacas (Vicugna paces) and one Llama (Lama glama) were immunized with the recombinant extracellular region of human NKp46. Subsequently, for each specimen a YSD library was constructed and selected by FACS. Sorting was conducted in a two-dimensional manner, to simultaneously detect for functional VHH surface expression as well as NKp46 binding. In this respect, approximately 10⁸ cells per library were sorted (Fig. IB, upper panel). For this, an antigen concentration of 1 pM was used, to also enrich for clones potentially displaying lower affinities. Intriguingly, for each of the libraries we already observed a distinct antigen-binding population of 2 - 4%. Subsequently, the sorting output was reanalyzed to get a glimpse about the enrichment, revealing more than 50% of antigen-binding cells in the FACS- sorted population (Fig. IB, lower panel). From each library 96 clones (288 clones in total) were sequenced and revealed a panel of 199 unique sdAbs. Based on clonotyping (a sequence identity of > 90% within CDR3 was defined as the same clonotype), we selected about 40 clones for bsAb expression (VHH NKp46.1 to VHH NKp46.41; for sequences of these VHHs and sequences of the CDR1, CDR2 and CDR3 of each of these VHHs, see table in section "Summary of Sequences"), each representing a unique clonotype (Fig. 1C).

In addition to sequence diversity of the VHH domains, most sequences revealed a considerable human-likeness, a low number of potential chemical degradation sites and post-translational modification sites as well as in general adequate in silica physico-chemical properties (Fig. 14). In more detail, sequence identity compared to the most similar human germline ranged between 63.4 % forNKp46.40 and 80.5 % for NKp46.1 if calculated for the complete sdAbs. Moreover, only a few sequences exhibited cysteines in non-canonical positions, such as NKp46.30, NKp46.36 and NKp46.38, all harboring two cysteines presumably forming an additional disulfide bond. Of note, only NKp46.37 displayed an unpaired cysteine residue that might potentially cause issues during manufacturing. Additionally, most of the screened VHHs revealed only a low number of amino acid residues considered as susceptible for potential (bio)chemical alterations, i.e., methionine oxidization, asparagine and glutamine deamidation as well as amino acid isomerization and 7V-glycosylation. Moreover, only two sequences, NKp46.32 and NKp46.33, showed considerable surface hydrophobicity (aggregation score). Finally, the set of identified clones displayed a broad coverage of computed isoelectric point (pl) values ranging from pH 3.2 up to pH 9.4. Due to these overall favorable developability properties determined in silico, these VHHs represent promising starting points for potential lead optimization studies.

For bsAb construction the strand-exchanged engineered domain (SEED) heterodimerization platform was applied that relies on beta-strand exchanges of IgG and IgA isotypes, resulting in preferential heavy chain heterodimerization (Davis et al., 2010). Each of the VHH domains was genetically engrafted onto the hinge region of the AG chain of the SEEDbody, whereas the Fab region of a humanized version of Cetuximab (hu225) was fused to the GA chain. The RF mutation was introduced into the GA chain in order to obviate purification of GA: GA homodimers that might form during expression (Tustian et al., 2016). Of note, an effector- silenced (eff-) version of the Fc region of the SEEDbody was used to solely focus on killing capacities mediated by the isolated NKp46-specific sdAb. Expression yields were in the triple digit milligram-per-liter scale for the vast majority of NKp46 SEEDbodies eff-, generally indicating adequate productivities for transient expression (Table I) (Pekar et al., 2021; Pekar et al., 2020). Besides, also aggregation propensities as determined by analytical size exclusion chromatography (SEC) post protein A purification indicated favorable biophysical properties of the herein engineered NKp46 SEEDbodies eff-. In this respect, SEC profiles for 37 out of 40 molecules were above 90% target monomer peak, except for NKp46.13 SEEDbody eff- with 87.1% target monomer peak, NKp46.20 SEEDbody eff- with 83.9% main peak purity and NKp46.29 SEEDbody eff- with 81.8% target monomer peak (Table I).

Initial binding experiments utilizing BLI at a NKp46 concentration of 100 nM revealed specific antigen binding for 30 out of the about 40 VHH-based NKCEs (Table I). Consequently, these 30 NKp46-specific SEEDbodies eff- were considered for further characterization. BLI was also exploited to analyze simultaneous binding to EGFR and NKp46. To this end, rhEGFR (ECD) was immobilized to the sensor tips, followed by a first association with the bispecific NKCE. Subsequently, a second association step was performed with the extracellular portion of rhNKp46 (Fig. 5). This unveiled simultaneous binding on the protein level for all NKp46- binding SEEDbodies eff-, whereas NKp46.37 SEEDbody eff-, which already did not show any binding to NKp46, also did not exhibit simultaneous binding behavior. Affinities with respect to NKp46 binding of VHH-based NKCE ranged from the lower double digit nanomolar range (NKp46.16, NKp46.17 and NKp46.31 SEEDbodies eff-) to binding in the sub-nanomolar range (NKp46.5, NKp46.10, NKp46.29 SEEDbodies eff-) with most of the molecules displaying affinities in the single digit nanomolar range (Table I).

Table I. Biochemical and biophysical properties of VHH-based NKCEs targeting NKp46 and EGFR.

Of note, non-binding molecules were excluded from this table. k_on is the rate constant of association, while k is the rate constant of dissociation. * Indicate the values for a second expression of the respective molecules

Example 3

Initial functional analysis

Initial functional analyses for the VHH-based NKCEs generated in Example 2 were conducted using the EGFR-overexpressing tumor cell line A431 as well as NK cells derived from PBMCs of seven healthy donors. Cetuximab was included as positive control, since it is known that this EGFR-directed antibody triggers very potent NK cell-mediated eradication of EGFR expressing tumor cells via ADCC. All compounds were assessed in terms of killing capacities at a concentration of 50 nM. Interestingly, while the extent of lysis differed noticeably between the studied molecules, all VHH-based NKCEs significantly triggered NK cell-dependent killing of A431 cells (Fig. 2A). Importantly, killing of EGFR-negative CHO cells was negligible, indicating tumor target-specific redirection of NK cells by the herein generated NKp46 SEEDbodies eff- (Fig. 6). Based on killing capacities, but also taking the sequence similarities of NKp46-directed paratopes into account, eleven NKCEs were selected for a more meticulous characterization. To this end, killing of A431 cells was evaluated in a dose-dependent manner using again PBMC-isolated NK cells of healthy donors as effector cells (Fig. 2B). As positive control, Cetuximab was again exploited, eliciting very potent NK cell-mediated killing (ECsokilling = 1.3 pM). For comparison we also utilized a monovalent (one armed) version of humanized Cetuximab, expressed as effector competent SEEDbody (oa_hu225 SEEDbody eff+). This molecule triggered lysis of A431 cells with a potency of 16.3 pM, whereas the same targeting arm in an effector-silenced Fc backbone (oa_hu225 SEEDbody eff-) was not capable of significantly inducing NK cell-dependent lysis of A431 cells. In contrast to this, all eleven selected VHH-based NKp46-specific NKCEs triggered dose-dependent NK-cell mediated lysis of A431 cells with potencies in the single digit picomolar to triple digit picomolar range. In this respect, NKp46.2 SEEDbody eff-, NKp46.18 SEEDbody eff-, and NKp46.21 SEEDbody eff- displayed the highest potencies with ECsokilling values in the low single digit picomolar range, clearly demonstrating robust killing capacities mediated by camelid-derived NKp46-targeting sdAbs.

Thus, the examined NKp46-specific VHH-based NKCEs elicit NK cell-mediated lysis of EGFR overexpressing tumor cells.

Example 4

Characterization of binding epitopes and NK cell activation activity

To further characterize the NKCEs and determine epitope specificities, pairwise competition for binding to NKp46 was performed for all eleven NKp46 SEEDbodies eff- in every possible combination (Fig. 3 A and Fig. 7A). For this, BLI experiments were conducted in which NKp46 was immobilized to the sensor tip, followed by two consecutive association steps utilizing distinct NKp46 SEEDbodies eff-. This revealed two groups sharing non-overlapping epitopes (epitope bin 1 for SEEDbodies eff- NKp46.2, NKp46.4, NKp46.8, NKp46.13, NKp46.15, NKp46.18, NKp46.21 and NKp46.25, and epitope bin 2 for NKp46.34 SEEDbody eff-). Interestingly, while consecutive binding was observed for NKp46.22 SEEDbody eff- and NKp46.34 SEEDbody eff- as well as NKp46.26 SEEDbody eff- and NKp46.34 SEEDbody eff- , successive binding was significantly diminished but still detectable for both clones and NKCEs that clustered to epitope bin 1. The same was true for competitive binding between both clones, NKp46.22 SEEDbody eff- and NKp46.26 SEEDbody eff-. Hence, it is tempting to speculate that clones within epitope bin 1, as well as SEEDbodies eff- NKp46.22 and NKp46.26 share overlapping, but distinct epitopes on NKp46 (Fig. 7B).

Subsequently, we examined NK cell activation mediated by the herein described NKp46- specific NKCEs in the presence of EGFR-overexpressing A431 cells. To this end, upregulation of CD69 as early NK cell activation marker was analyzed for NKp46.2 SEEDbody eff- as representative clone of epitope bin 1 as well as NKp46.22 SEEDbody eff-, NKp46.26 SEEDbody eff- and NKp46.34 SEEDbody eff-, each targeting a unique epitope on NKp46 (Fig. 3B, Fig. 8). All four NKCEs triggered significant activation of NK cells compared to the Fc effector-silenced one-armed EGFR-targeting negative control (oa_hu225 SEEDbody eff-). Interestingly, NK cell activation was slightly lower for all NKCEs triggering NKp46 than for Cetuximab as well as compared with a monovalent Fc effector competent humanized version of Cetuximab, both mediating NK cell activation via FcyRIIIa ligation, clearly highlighting the impact of FcyRIIIa as very potent trigger molecule for NK cell activation.

Thus, the examined NKp46-specific sdAb-based NKCEs target distinct epitopes and mediate significant NK cell activation.

Example 5

Effects of co-engagement of NKp46 and FcyRIIIa

It has been described before that killing capacities of NKp46-directed EGFR-targeting bispecific NKCEs can be further enhanced by co-triggering FcyRIIIa (Pekar et al., 2021; Klausz et al., 2022). This was also demonstrated by Vivier and colleagues for NKp46-specific Fab- derived paratopes incorporated into multifunctional NKCEs (Gauthier et al., 2019). To investigate, if this also holds true for sdAb-derived NKCEs specific for NKp46, SEEDbodies NKp46.2 and NKp46.26 were expressed harboring an effector function enabled Fc region (eff+). These two sdAb-based paratopes were chosen because of differences in affinities, epitope targeting and initial killing capacities (Table I, Fig. 2 and Fig. 3A, Fig. 7). Intriguingly, co-engagement of FcyRIIIa augmented both, potencies as well as efficacies (maximal lysis) for each studied NKCE (Fig. 4A). In this respect, for NKp46.2 SEEDbody a moderate enhancement in ECsokilling was observed (ECso NKp46.2 SEEDbody eff- of 0.98 pM v ECso NKp46.2 SEEDbody eff+ of 0.61 pM). This effect was even more pronounced for NKp46.26 SEEDbody, where potencies were augmented by a factor of seven (ECso NKp46.26 SEEDbody eff- of 4.1 pM v EC50 NKp46.26 SEEDbody eff+ of 0.59 pM), corroborating the impact of the Fc backbone for NKCEs targeting NCRs.

Thus, the killing capacities of the NKp46-targeting NKCEs can be augmented by coengagement with other NK cell activating receptors like FcyRIIIa.

Example 6

NKp46-specific VHH-based NKCEs with further engineered antibody formats

In this example, the influence of valencies for each paratope on tumor cell lysis as well as the impact of the overall antibody design architecture was investigated (Fig. 4B). To this end, three additional formats were employed, all harboring an effector-silenced Fc region (Fig. 4B, Design architecture B-D) as well as either VHHs NKp46.2 or NKp46.26. While initially, all molecules were tested harboring strictly monovalent N-terminally linked paratopes (Fig. 4B, Format A), subsequently also molecules allowing for bivalent targeting of NKp46 were produced. For this, the respective VHHs were engrafted in a tandem arrangement separated by a 20 amino acid Gly/Ser linker and fused to the N-terminus of the AG chain of the SEED, while on the GA chain the Fab arm of hu225 was utilized (Format B). In addition, the respective VHH was fused to the C-terminus of an effector-silenced IgGl derivative of hu225, enabling bivalency for both paratopes (Design C; of note a 20 amino acid Gly/Ser linker was implemented between the Fc part and the VHH). Finally, fusion of the Fab arm of hu225 to both N-termini of the SEED chain as well as engraftment of only one respective VHH to the C-terminus of the AG chain of the SEED (separated by a 20 amino acid Gly/Ser linker) enabled bivalent targeting of EGFR, as well as addressing Nkp46 in a monovalent fashion (Format D).

In general, all different formats could successfully be produced in Expi293 cells with expression yields in the double to triple digit milligram per liter scale (Table II). Interestingly, slightly reduced expression yields were observed for both NKp46-specific sdAbs when engrafted in asymmetric design architecture D (double digit mg/L vs triple digit mg/L), suggesting an overall reduced productivity for this particular format. Besides, molecules representing different antibody architectures unveiled a high purity after expression and affinity purification. Except for NKp46-specific VHH NKp46.26 in format A (showing a slightly broader target peak with 86.9% purity), size exclusion chromatography (SEC) profiles showed target monomer peak purities of above 90% (Table II, Fig. 9). Also, thermal stabilities were quite similar between the different formats for a given engrafted VHH paratope, i.e., Tml ranging from 64.1 °C to 68.5 °C for NKp46.2 and 54.9 °C to 56.0 °C forNKp46.26, with VHHNKp46.2 in format C (IgGl- based format) that seemed to be the most stable design exhibiting its first unfolding transition midpoint at 68.5 °C. Additionally, a lower overall thermostability for all NKp46.26 harboring molecules was found (Table II, Fig. 10). Furthermore, employed HIC analysis was employed in order to determine the relative hydrophobicity of the generated NKCE architectures (Table II, Fig. 11). Therefore, as reference two therapeutic antibodies were used, Cetuximab and Avelumab (HIC retention times of 5.8 min and 7.2 min, respectively), that were granted marketing approval by the FDA. In general, a trend towards higher retention times and hence for higher hydrophobicity was observed in cases where more paratopes were incorporated into a given molecule and for the designs where the NKp46 VHHs were fused to the C-terminus of the molecule (Design C and D). In this respect, HIC retention times were 6.8 and 6.5 minutes, respectively, for molecules NKp46.2 C and NKp46.26 C that harbor four paratopes in total. In contrast to this, HIC retention times were 6.1 min and 5.8 min when both paratopes were engrafted into the strictly monovalent format A. Consequently, the NKCE architectures incorporating three paratopes, i.e., design B and D, showed intermediary HIC retention times with NKp46.2 B and NKp46.26 B eluting after 6.3 and 5.8 minutes as well as NKp46.2 D and NKp46.26 D eluting after 6.6 and 6.5 minutes, respectively. Overall, HIC retention times of all different molecules were between those of Cetuximab and Avelumab, clearly demonstrating adequate biophysical properties for herein engineered NKCE architectures.

Table II. Biophysical and functional properties of different VHH-based EGFR x NKp46 NKCE format architectures.

Furthermore, the ability of the different NKCE formats to redirect NK cell cytotoxicity against EGFR-overexpressing A431 cells was investigated. Similar to the initially observed results in format A (Fig. 2B), both NKp46-directed VHHs triggered lysis in the low picomolar range (EC₅₀NKp46.2 of 0.98 pM, EC₅₀NKp46.26 of 4.1 pM; Fig. 4A, C and D). Bivalent targeting of activating receptor NKp46 on the NK cell in format B augmented killing capacities for both engrafted VHHs. Intriguingly, for VHH NKp46.2 not only a slightly enhanced potency in format B was observed (EC50 NKp46.2 B of 0.59 pM v EC50 NKp46.2 A of 0.98 pM) but also a trend towards higher efficacies, i.e., maximum lysis (Fig. 4C). For NKp46.26 the effect was even more distinct in terms of potencies (EC50 NKp46.26 B of 0.60 pM vs EC50 NKp46.26 A of 4.1 pM), resulting in an improvement of approximately 7-fold. However, bivalent targeting of NKp46 had no impact on maximum killing capacities (Fig. 4D). This is in strong contrast to the design architectures that allow for bivalent targeting of EGFR (Fig. 4 C and D, format C & D). Here, maximum killing capacities were significantly enhanced compared to both formats that only enable monovalent targeting of the tumor associated antigen. Though, for VHH NKp46.2 no significant benefit was observed in terms of potencies (EC50 NKp46.2 C of 1.08 pM vs EC50 NKp46.2 A of 0.98 pM). In fact, potencies seem to be even reduced when reformatted into format D, allowing for bivalent targeting of EGFR and monovalency for NKp46 (EC50 NKp46.2 D of 4.67 pM v EC50 NKp46.2 A of 0.98 pM; yet, this is partially misleading, since significantly enhanced efficacies result in limited comparability). Opposed to these findings, for VHH NKp46.26 bivalent targeting of EGFR and for NKp46 resulted in both, enhanced potencies by the factor of approximately 6-fold (EC50 NKp46.26 C of 0.67 pM vs EC50 NKp46.26 A of 4.1 pM) as well as significantly augmented maximal killing capacities (Fig. 4D). Even in design D reformatting of this particular sdAb largely maintained potencies albeit displaying much higher efficacies. Importantly, for all molecules analyzed killing of EGFR-negative CHO cells was negligible (Fig. 12). Ultimately, this shows that killing capacities of VHH-derived NKp46-specific NKCEs can be further augmented by antibody format engineering. Thus, Antibody format engineering enables the generation of NKp46-specific VHH-based NKCEs with further enhanced potencies and efficacies.

Example 7

Humanized VHH1 Variants

All methods in Example 7 are carried out as described in Example 1.

Humanized variants of the VHHs as listed in the Table of VHHs are prepared. In these humanized versions, the CDRs are identical to those of the respective "parent" VHH, but individual amino acids in the framework regions and/or the CDRs are substituted compared to the parent VHH by corresponding amino acids of a human germline sequence.

Constructs are expressed in small scale expression as described in Experiment 1. After purification (identity confirmed by mass validation), purified variants are analyzed by size exclusion chromatography (SEC) and their binding to NKp46 and KD value is determined by biolayer interferometry (BLI) measurements as described in Experiment 1.

From these data, it can be seen that the prepared humanized variants show in vitro properties that are similar to the parent VHHs in terms expression yield/purity, KD, binding capacity, and thus expected functionality.

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Claims

A compound comprising a VHH antibody domain or a fragment thereof, wherein

(a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH NKp46.1, VHH NKp46.2, VHH NKp46.3, VHH NKp46.4, VHH NKp46.5, VHH NKp46.6, VHH NKp46.7, VHH NKp46.8,

VHH NKp46.9, VHH NKp46.10, VHH NKp46.11, VHH NKp46.12, VHH

NKp46.13, VHH NKp46.14, VHH NKp46.15, VHH NKp46.16, VHH

NKp46.17, VHH NKp46.18, VHH NKp46.20, VHH NKp46.21, VHH

NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH

NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29, VHH

NKp46.31 and VHH NKp46.34 as shown in the Table of CDRs below;

Table of CDRs:

163

164

2. A compound comprising a VHH antibody domain or a fragment thereof, wherein

NKp46.22, VHH NKp46.23, VHH NKp46.24, VHH NKp46.25, VHH

NKp46.26, VHH NKp46.27, VHH NKp46.28, VHH NKp46.29, VHH

NKp46.31 and VHH NKp46.34 as shown in the Table of VHH Sequences below;

165 (B) said VHH antibody domain comprises a VHH sequence as defined in (A) with modification, wherein the modification is that said sequence is humanized;

Table of VHH Sequences:

166

3. The compound according to any one of claims 1 or 2, wherein said compound binds to NKp46 with a KD of l >< 10'⁶ M or stronger in a binding assay by BLI (biolayer interferometry) with recombinant extracellular domain of NKp46 (NKp46-ECD, SEQ ID NO: 165) in KB buffer (PBS + 0.1 % Tween-20 + 1% BSA).

4. The compound according to any one of claims 1 to 3, wherein said compound further comprises a targeting moiety.

5. The compound according to any one of claims 1 to 4, wherein said targeting moiety is capable of specifically binding to a tumor-associated antigen.

6. The compound according to any one of claims 1 to 5, wherein said compound comprises an antibody Fc region competent in Fc receptor binding.

7. A pharmaceutical composition comprising the compound according to any one of claims 1 to 6.

8. The compound according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7 for use as a medicament.

169