VHH-based NKP30 binder

Abstract

The present disclosure relates to VHH-based NKp30 binders having preferred characteristics. Furthermore, the present disclosure relates to pharmaceutical compositions comprising such compounds, and to the use of such compounds and such pharmaceutical compositions in methods of medical treatment.

Description

[Technical field]

[0001] FIELD OF THE PRESENT APPLICATION The present disclosure relates to VHH-based NKp30 binders having preferred characteristics. Furthermore, the present disclosure relates to pharmaceutical compositions comprising such compounds, and to the use of such compounds and such pharmaceutical compositions in methods of medical treatment. [Background technology]

[0002] 2. Background of the Invention Despite advances in clinical treatments over the past decades, cancer remains one of the leading causes of death in the developed world. In recent years, novel approaches to harness the power of the immune system have been expected to provide considerable progress, particularly by activating immune cells in the body and directing them against tumor cells. One cell type that shows great potential in this regard is the natural killer cell (NK cell).

[0003] NK cells play a central 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, which recognize distinct antigens via variable T cell receptors, the discrimination between healthy and stressed cells, and the resulting antitumor response of NK cells, is based on a sophisticated intercommunication between numerous germline-encoded activating and inhibitory receptors (Gonzales-Rodriguez et al., 2019; Chiossone et al., 2018).

[0004] Natural killer cells are innate lymphoid cells that recognize discontinuities and dangers in multiple tissue compartments by integrating positive and negative signals. Negative signals are generally mediated by interactions 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 delivered through interactions with their ligands, including an array of NK activating receptors (Natural Cytotoxicity Receptors, NCRs; NKp30, NKp46, NKp44), NKG2D, and DNAM-1, as well as costimulatory molecules including 4-1BB (Koch et al., 2017; Morgado et al., 2011). For NCR and NKG2D, many of the ligands are "danger signals" that are upregulated on stressed and diseased tissues, including virus-infected cells and tumor cells. Another mechanism by which NK cells are activated is the bridging of low-affinity activating FcγRIIIa (CD16a) on NK cells with cells opsonized with IgG or bispecific antibodies. Unlike NCR and NKG2D, signaling through FcγRIIIa is often more robust in resting NK cells, but is modulated by multiple variables, including functionally distinct polymorphic variants of FcγRIIIa as well as competition for binding with circulating IgG. Ultimately, the balance between activating and inhibitory signals determines whether NK cells are activated. Therefore, NK cells have an intrinsic ability to discriminate between healthy and diseased tissues.

[0005] Ultimately, activation of NK cells leads to lysis of target cells via degranulation, i.e., release of cytotoxic substances such as perforin and granzymes, and production of pro-inflammatory cytokines and chemokines. NK cells have shown great potential in the treatment of cancer by various approaches.

[0006] Several early clinical trials employing adoptive transfer of wild-type or genetically modified (e.g., CAR) NK cells, alone or in combination with antibodies, as a cancer treatment modality have shown promising early results for hematological malignancies ( Gonzales-Rodriguez et al., 2019 ; Burger et al., 2019 ; Rezvani et al., 2019 ).

[0007] Although adoptive cell therapy with ex vivo activated NK cells represents a promising approach, its logistical complexity has also driven the development of NK-directed antibody-based approaches for cancer immunotherapy. In this regard, 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 that allow 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 Fcγ receptor CD16a. CD16a-ligation of antibodies bound to their target cells induces potent NK cell degranulation (Bryceson et al., 2005). This process is termed antibody-dependent cellular cytotoxicity (ADCC) and is considered one of the key mechanisms of action of many therapeutic antibodies (Seidel et al., 2013).

[0008] However, the ADCC-inducing ability of antibodies is affected by antigen density on target cells. Due to the low affinity interaction of antibodies with CD16a, low antigen density typically results in mild opsonization and consequently limited induction of ADCC (Koch et al., 2017). Moreover, CD16a polymorphisms have been described in humans, resulting in different levels of ADCC depending on the patient's genotype. Finally, conventional therapeutic antibodies must compete with serum immunoglobulins for binding to CD16a, resulting in limited CD16a occupancy and limited ADCC potency (Ellwanger et al., 2019).

[0009] To overcome these drawbacks inherent to classical antibody therapy, bi- and multispecific NK cell engagers have been developed, where one paratope binds with high affinity to the activating receptor CD16a, while the other is directed against a tumor-associated antigen (Koch et al., 2017; Rothe et al., 2015). In 2019, Vivier and colleagues described the efficient generation of a trifunctional NK cell engager (Gauthier et al., 2019). In their study, the authors employed two activating receptors of NK cells, NKp46 as well as CD16 (Fc-mediated), for effector cell engagement. In a direct comparison with rituximab and Fc-modified obinutuzumab in in vivo studies in mice, the developed NK cell engager further supported the opinion that this class of molecules could be a promising therapeutic entity for tumor treatment.

[0010] Alternatively, natural or synthetic ligands of NK cell receptors can be combined with tumor targeting moieties in a bispecific format, thus constructing effector cell engagers. Such bispecific or trifunctional entities that form a bridge between activating receptors on NK cells and tumor-associated antigens (TAA) on tumor cells are referred to as NK cell engagers (Koch et al., 2017). Bispecific antibodies targeting TAA (e.g., CD20) and NKp46, NKG2D and NKp30 (Peipp et al., 2015; Kellner et al., 2016) have demonstrated potent target-dependent cytotoxicity and cytokine release in vitro, either through the antibody moiety or recombinant forms of the ectodomain of the ligand (e.g., ULBP2) (von Strandmann et al., 2006).

[0011] NKp30 is an activating receptor expressed on the majority of NK cells. Its cell-binding ligand B7-H6 is upregulated on tumor cells and absent on most normal cells. Another less well-characterized ligand is HLA-B-associated transcript 3 (BAT3) / Bcl2-associated athanogene 6 (BAG6), which is expressed in the nucleus and can be transported to the plasma membrane or released into exosomes. Importantly, reduced NKp30 expression correlated with reduced survival in AML, and fewer NK cells expressing NKp30 were found in patients with gastric or breast cancer compared to healthy donors. Taken together, these data suggest that the NKp30-receptor axis may play an important role in tumor surveillance of various tumor entities. Potential strategies to modulate the NKp30 axis may therefore represent a promising approach to promote antitumor NK cell responses.

[0012] Despite the great potential of NK cell engagers, their use in practice is limited because available activating ligands for NK cells do not have a suitable profile of biophysical and / or functional characteristics (e.g., they have insufficient affinity for effective NK cell activation, cannot be produced economically in large quantities, do not have good stability when exposed to heat and solvents, and / or are not amenable to genetic engineering for all desired uses (such as scaffolding, labeling, and modifications to specific amino acids)).

[0013] Consequently, there is a need in the art for improved methods of cancer treatment. Moreover, there is a need in the art for improved methods of NK cell activation. Moreover, there is a need in the art for compounds that activate NK cells, particularly compounds that activate NK cells and bind to NKp30, with improved characteristics such as improved affinity, improved specificity, improved stability, improved manufacturability, improved amenability to genetic manipulation, improved potency and / or efficacy for killing tumor cells, increased efficacy in releasing pro-inflammatory cytokines, improved pharmacokinetics, reduced side effects, increased therapeutic window, and / or increased patient safety. Moreover, there is a need in the art for compounds that activate NK cells, particularly compounds that activate NK cells and bind to NKp30, that are not affected by the presence of the natural B7-H6 ligand. Moreover, there is a need in the art to address the above needs with a "standardized" approach that can be widely used to activate NK cells (e.g., in combination with various targeting moieties) and / or allows for inexpensive and rapid synthetic access. The present disclosure overcomes the above problems and addresses the above needs. Summary of the Invention

[0014] SUMMARY OF THE PRESENT APPLICATION The present disclosure addresses the needs described above in the "Background of the Invention" section by various aspects and embodiments described below.

[0015] The present invention is based in part on the surprising observation that compounds comprising a VHH antibody domain or fragments thereof as described in the present disclosure exhibit a combination of various advantageous effects.For example, the advantageous effects may include (but are not limited to) high affinity for NKp30, high K on rate for NKp30 binding, low K off rate for NKp30 binding, high efficiency in activating NK cells, high efficiency in inducing cytokine release (interferon-γ, TNF-α), (particularly in the context of molecules, also including targeting moieties), enhanced cytotoxicity (e.g., in terms of potency and / or efficacy), and improved manufacturability.When used in combination with an Fc region capable of FcγRIIIa binding, this cytotoxicity is further enhanced.

[0016] In one aspect, the present disclosure relates to a compound comprising a VHH antibody domain or a fragment thereof, (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2, and CDR3 of one of VHH1 to VHH16 as shown in the CDR table below; (b) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with a modification, wherein the modification is that at least one sequence 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 a modification, wherein the modification is - substitutions, additions or deletions of up to 3 amino acids in CDR1, - replacement, addition or deletion of up to 3 amino acids in CDR2, and / or - substitutions, additions, or deletions of up to 3 amino acids in the CDR3; CDR Table: [Table A-1]

[0017] [Table A-2]

[0018] In another aspect, the present disclosure relates to a compound comprising a VHH antibody domain or a fragment thereof, (A) the VHH antibody domain comprises any one of VHH1 to VHH16 shown in the VHH sequence table below; (B) the VHH antibody domain comprises a VHH sequence as defined in (A) with a modification, wherein the modification is that the sequence is humanized; (C) the VHH antibody domain comprises a VHH sequence as defined in (A) with a modification, wherein the modification is a replacement, addition or deletion of up to 25 amino acids; or (D) the VHH antibody domain comprises a VHH sequence that is at least 75% identical to the VHH sequence set forth in (A); Table of VHH sequences: [Table B-1]

[0019] [Table B-2]

[0020] In another aspect, the present disclosure relates to a compound comprising a VHH antibody domain or a fragment thereof, (A) the VHH antibody domain consists of any one of VHH1 to VHH16 shown in the VHH sequence table below; (B) the VHH antibody domain consists of a VHH sequence as defined in (A) with a modification, wherein the modification is that the sequence is humanized; (C) the VHH antibody domain consists of a VHH sequence as defined in (A) with a modification, wherein the modification is a substitution, addition or deletion of up to 25 amino acids; or (D) The VHH antibody domain consists of a VHH sequence that is at least 75% identical to the VHH sequence mentioned in (A).

[0021] In another aspect, the present disclosure relates to a pharmaceutical composition comprising a 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.

[0022] In another aspect, the present disclosure relates to a method of treating a disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound according to the present disclosure or a pharmaceutical composition according to the present disclosure.

[0023] In another aspect, the present disclosure relates to the use of a compound according to the present disclosure or a pharmaceutical composition according to the present disclosure for the manufacture of a medicament, preferably for the treatment of a disease or disorder as defined below. [Brief description of the drawings]

[0024] Brief description of the figure The following references are made to the figures: All methods referred to in the description below the figures were carried out as detailed in the Examples. [Figure 1A-B]Figure 1. Generation of NKCE (NK cell engager) based on NKp30-specific VHH single domain antibodies by camelid immunization combined with yeast surface display (YSD). (A) Scheme depicting the generation of NKp30-targeting VHHs utilized to construct bispecific NKCE. After immunization of three camelids with recombinant human NKp30, YSD was employed for the isolation of antigen-specific VHHs. The VHHs were then assembled into bispecific SEED bodies harboring humanized Fab arms of cetuximab for tumor targeting. For in-depth characterization, Fc-mediated effector functions were silenced by introducing specific point mutations in the Fc region. (B) FACS selection for the isolation of NKp30-specific VHHs by implementing full-length VHH display and a two-dimensional staining strategy for NKp30 binding. Of note, in sorting round 1, all three sub-libraries based on each immunized specimen were sorted separately (only the sorting of the llama-derived sub-library is shown), and then the output of sorting 1 was combined for the subsequent sorting round 2. [Figure 1C-D]Figure 1. Generation of NKCE (NK cell engager) based on NKp30-specific VHH single domain antibodies by camelid immunization combined with yeast surface display (YSD). (A) Scheme depicting the generation of NKp30-targeting VHHs utilized to construct bispecific NKCE. After immunization of three camelids with recombinant human NKp30, YSD was employed for the isolation of antigen-specific VHHs. The VHHs were then incorporated into bispecific SEED bodies harboring humanized Fab arms of cetuximab for tumor targeting. For in-depth characterization, Fc-mediated effector functions were suppressed by introducing specific point mutations in the Fc region. (B) FACS selection for the isolation of NKp30-specific VHHs by implementing full-length VHH display and a two-dimensional staining strategy for NKp30 binding. Of note, in sorting round 1, all three sub-libraries based on each immunized specimen were sorted separately (only the sorting of the llama-derived sub-library is shown), and then the output of sorting 1 was combined for the subsequent sorting round 2.

[0025] [Figure 2A] FIG. 2 shows the results obtained in biolayer interference experiments studying the competition of VHH SEED bodies with B7-H6 or between different VHH SEED bodies for binding to NKp30. [Figure 2B] FIG. 2 shows the results obtained in biolayer interference experiments studying the competition of VHH SEED bodies with B7-H6 or between different VHH SEED bodies for binding to NKp30.

[0026] [Diagram 3]Figure 3 shows data from chromium release experiments that lead us to the conclusion that EGFR-positive tumor cells were potently killed by NKp30 VHH SEED bodies. Standard 4h chromium release assays were performed on high EGFR expressing A431 cells (left graph) and lower EGFR expressing A549 cells (right graph) using MNCs from healthy donors at an effector-to-target cell (E:T) ratio of 80:1 and increasing concentrations of VHH SEED bodies competing (red) and not competing with B7-H6 (green / brown). One-armed SEED bodies lacking NKp30 VHH single domain antibodies (oa_hu225-SEED bodies) and the monoclonal antibody cetuximab (black squares) were used as controls and for comparison. Mean values ​​± SEM of three independent experiments in triplicate are shown. The leading candidates for further analysis are bolded.

[0027] [Figure 4A] Figure 4 shows an experiment to study tumor cell lysis and cytokine release by VHH SEED bodies. VHH SEED bodies competing with B7-H6 (eff-) show improved lysis of tumor cells compared to non-competing VHH SEED bodies (eff-), whereas all NKCES induce a strong cytokine release from activated NK cells. (A) A standard 4h chromium release assay was performed on A431 cells (left graph) and A549 cells (right graph) using isolated NK cells of a healthy donor at an E:T ratio of 10:1 and increasing concentrations of VHH SEED bodies competing (red) and not competing with B7-H6 (green / brown). A one-arm SEED body lacking the NKp30 VHH single domain antibody (oa_hu225-SEED body) and the monoclonal antibody cetuximab (black squares) were used as controls and for comparison. Mean values ​​± SEM of three independent experiments in triplicate are shown. [Figure 4B](B) Cytokine release induced by selected VHH SEED bodies (eff-) at saturating concentrations. NKCE and cetuximab were compared to oa_hu225 SEED bodies for promoting NK cell-mediated IFN-γ and TNF-α release using a cytokine HTRF kit for quantification. Purified NK cells were co-cultured with A431 cells at an E:T ratio of 5:1 for 24 h prior to analysis. Graphs show box plots overlaid with dot plots of seven individual experiments, respectively. ***p is 0.001, **p is 0.01, *p is 0.05. ns is not significant compared to oa_hu225 SEED bodies.

[0028] [Diagram 5] Figure 5 shows data from an experiment confirming that EGFR-positive tumor cell killing with VHH SEED bodies is not compromised by high concentrations of soluble B7-H6. Standard 4h chromium release assays were performed in A431 (left graph) and A549 cells (right graph) using NK cells from healthy donors at an E:T ratio of 10:1 and saturating concentrations of SEED bodies in the presence (grey bars) or absence (black bars) of B7-H6 ECD. One-arm SEED bodies lacking NKp30 VHH single domain antibodies (oa_hu225-SEED bodies), monoclonal antibody cetuximab, and B7-H6 ECD alone served as controls. Mean values ​​± SEM of four independent experiments are shown. VHH1 = competitor of B7-H6, VHH2 = non-competitor of B7-H6, VHH4 = partial competitor.

[0029] [Figure 6]Figure 6 shows data from an experiment confirming that effector-functional Fc improves EGFR-positive tumor cell killing by non-competing VHH SEED bodies. Standard 4h chromium release assays were performed on A431 (left graph) and A549 (right graph) cells using isolated NK cells from healthy donors at an E:T ratio of 10:1 and increasing concentrations of B7-H6-competing VHH1 SEED bodies (orange) and non-competing VHH2 SEED bodies (green) harboring either effector-functional (eff+; solid line, filled symbols) or non-functional Fc (eff-; dotted line, open symbols). One-arm SEED bodies lacking the NKp30 VHH single domain antibody but with effector-functional (oa_hu225-SEEDbody eff+) or non-functional (oa_hu225-SEEDbody eff-) Fc, as well as the monoclonal IgG1 antibody cetuximab (black squares) were used as controls and for comparison. Mean values ​​± SEM of four independent experiments in triplicate are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Overview of Arrays [Table C-1]

[0031] [Table C-2]

[0032] [Table C-3]

[0033] [Table C-4]

[0034] [Table C-5]

[0035] [Table C-6]

[0036] [Table C-7]

[0037] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Although the present disclosure is described in detail above and below, it should be understood that the present disclosure is not limited to the specific methodology, protocols, and reagents described by the present disclosure, as they may vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to limit the scope of the present disclosure, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0038] In the following, certain elements of the present disclosure, including the description of specific embodiments, will be described in more detail. However, the various described examples and preferred embodiments should not be interpreted to limit the present disclosure to only the embodiments explicitly described. This description should be understood to support and cover the embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements in any manner. Furthermore, any permutation and combination of all elements described in this application should be considered as disclosed by the description of this application, unless this leads to logical inconsistency or the context indicates otherwise.

[0039] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings that are commonly understood by those skilled in the art.Generally, the nomenclature and techniques mentioned in this disclosure, for example, the nomenclature and techniques of organic chemistry, chemical synthesis, biology, medicinal and pharmaceutical chemistry, medicine, pharmacology, or toxicology, are well known and commonly used in the art.The methods and techniques of this disclosure are generally carried out according to conventional methods well known in the art and as described in the cited references, and are discussed throughout this disclosure unless otherwise indicated.

[0040] According to a first aspect, the present disclosure relates to a compound comprising a VHH antibody domain or a fragment thereof, (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2, and CDR3 of one of VHH1 to VHH16 as shown in the CDR table below; (b) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with a modification, wherein the modification is that at least one sequence 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 a modification, wherein the modification is - substitutions, additions or deletions of up to 3 amino acids in CDR1, - replacement, addition or deletion of up to 3 amino acids in CDR2, and / or - substitutions, additions, or deletions of up to 3 amino acids in the CDR3; CDR Table: [Table D-1]

[0041] [Table D-2]

[0042] "Compound" as used in this disclosure is not specifically limited and refers to any chemical entity of any chemical class, but includes protein domains as defined above. Thus, a compound may be, for example, an organic compound, or a compound composed of organic and inorganic parts, a protein composed of a single amino acid chain, a protein composed of multiple amino acid chains associated either non-covalently or covalently, or a non-covalent complex that includes an inorganic component. A compound may consist of the amino acid sequence of a VHH antibody domain or a fragment thereof alone, or may additionally include additional amino acid(s) that may be covalently or non-covalently attached, or may be associated with an inorganic component. Preferably, a compound is a molecule.

[0043] For example, the compound can be a bispecific molecule comprising a VHH antibody domain according to the present disclosure covalently linked to an IgG1 antibody lacking one of its "arms. " Alternatively, the compound can be an antibody with a VHH antibody domain according to the present disclosure and a targeting moiety prepared in a SEED format, resulting in a bispecific antibody with the structure shown in Figure 1.

[0044] As the skilled artisan will appreciate, many other formats of compounds are possible, provided the resulting compound does not interfere with the function of the VHH antibody domain or its fragment, i.e., binding to NKp30 and activation of NK cells.

[0045] The compounds of the present disclosure can be prepared by standard methods of genetic engineering and recombinant protein technology known to those of skill in the art (see, for example, 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.

[0046] In cases where the compounds cannot be expressed alone, the individual portions can be prepared separately and later covalently coupled, for example, either by chemical reaction at suitable reactive groups (e.g., by maleimide chemistry) or by enzymatic ligation (e.g., by transglutaminase catalyzed ligation). For example, a VHH antibody domain or fragment thereof can be prepared by recombinant protein expression followed by ligation to an antibody or antibody fragment, resulting in a bispecific antibody compound as described in the Examples section.

[0047] Where the compounds contain non-biomolecular (such as peptidomimetics or small molecules) building blocks, these building blocks may, for example, be obtained by standard methods of synthetic organic chemistry.

[0048] An "antibody" is a polypeptide substantially encoded by an immunoglobulin gene or genes, or an antigen-binding fragment thereof, that specifically binds to 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.

[0049] In primates, such as humans, the variable domains of the heavy and light chains of an antibody combine to specifically bind to an antigen. Generally, naturally occurring primate (e.g., human) or mouse immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). There are five major classes (or isotypes) of heavy chains that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Primate antibodies can be class switched.

[0050] Certain IgG antibodies from members of the camel and dromedary (Camelus bactrianus and Calelus dromaderius) families, including newer global members such as the mammalian llama species (Lama paccos, Lama glama, and Lama vicugna), lack light chains as found in nature and are therefore structurally distinct from the typical four-chain quaternary structure with two heavy chains and two light chains for antibodies from other animals. See PCT / EP93 / 02214 (WO 94 / 04678 published March 3, 1994). Such llama IgG subtypes lack light chains and CH1 domains and are called heavy chain antibodies. These naturally occurring camelid antibodies, consisting only of heavy chains, are functional and stable even in the absence of light chains. The antigen-binding site of these heavy chain antibodies is formed only by a single domain, termed "VHH" (Koenning et al., 2017), which is therefore used synonymously herein as "VHH antibody domain."

[0051] Each light and heavy chain of an antibody contains a constant region and a variable domain. Reference to "VH" or "VH" refers to the variable region of an immunoglobulin heavy chain, including fragments of that antibody. Reference to "VL" or "VL" refers to the variable region of an immunoglobulin light chain, such as that in a primate antibody. The variable domain of a heavy chain antibody is called a VHH. VHH consists of only one polypeptide chain of 15 kDa and is considered to be the smallest known naturally occurring domain with full antigen-binding capacity.

[0052] The variable domains of the light and heavy chains contain a "framework" region interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs" (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, USDepartment 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, 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.

[0053] CDRs are typically referred to as CDR1, CDR2, and CDR3 (from 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 L1, CDR L2, and CDR L3. Heavy chain CDRs are sometimes referred to as CDR H1, CDR H2, and CDR H3. VHH monoclonal antibodies only have a heavy chain, and therefore only include one CDR1, CDR2, and CDR3. Generally, CDR3 is primarily responsible for antigen specificity.

[0054] VHHs comprise, in N- to C-orientation, the following structural regions: N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C, where FR denotes the amino acid sequences of the framework regions and CDR denotes the amino acid sequences of the complementarity determining regions (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, 1991).

[0055] The extents of the framework regions and CDRs have been identified (see Kabat et al., Sequences of Proteins of Immunological Interest, USDepartment 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 that include a light chain (such as primate antibodies), 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 currently maintained online. The locations of camelid CDRs can also be determined (see, e.g., Sircar et al., J. Immunol. 186:6357-6367, 2011); the RosettaAntibody program, a program for determining camelid antibody structure, is available on the internet.

[0056] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by a cell transfected with a single antibody heavy chain gene (and optionally a light chain gene, such as a primate antibody). Monoclonal antibodies may be obtained using a variety of techniques known to those skilled in the art, including standard hybridoma technology (see, for example, Koehler 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, for example, expression from a eukaryotic host cell transfected with a DNA molecule encoding the homogeneous antibody or expression from a prokaryotic host cell transfected with a DNA molecule encoding the homogeneous antibody.

[0057] VHH antibody domains can be obtained by genetic engineering to produce small proteins with high affinity to the target, resulting in low molecular weight antibody-derived proteins. See, for example, Sellmann et al., 2020; U.S. Patent No. 5,759,808 issued June 2, 1998; also see Dumoulin et al., (2003); Pleschberger et al., (2003); Cortez-Retamozo et al., (2002); and Lauwereys et al., (1998).

[0058] In some embodiments, VHH molecules can be produced as recombinant monoclonal antibodies or antigen-binding fragments in various expression platforms that avoid the use of hybridomas and mice. The VHH monoclonal antibodies can be humanized monoclonal antibodies. In some embodiments, the monoclonal antibodies can be chimeric antibodies. Without being bound by theory, VHH monoclonal antibodies have approximately one tenth the molecular weight of a human IgG molecule, and the protein has a physical diameter of only a few nanometers.

[0059] One consequence of their small size is that VHH monoclonal antibodies can bind to antigenic sites that are functionally invisible to larger antibody proteins, making them useful as reagents for detecting cryptic antigens using classical immunological techniques and thus serving as therapeutic agents. Thus, another consequence of their small size is that camelid VHH monoclonal antibodies can inhibit as a result of binding to specific sites in grooves or narrow clefts of target proteins and therefore act with a force closer to the function of classical drugs of low molecular weight than classical antibodies.

[0060] Without being bound by theory, the low molecular weight and compact size further result in camelid VHH monoclonal antibodies that are extremely thermally stable, stable to extreme pH and proteolytic digestion, and poorly antigenic. Furthermore, these molecules can be fully expressed in prokaryotic cells such as E. coli, and are expressed and functional as fusion proteins with bacteriophages.

[0061] "Humanized" antibody / antibody sequence, as used herein, refers to the process of adapting a non-human (such as camelid, llama, or synthetic) antibody sequence to be more similar to a human antibody sequence by replacing one or more individual amino acids with the corresponding amino acid of the human antibody sequence. Typically, a 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, for example, by a BLAST search. Corresponding amino acids can then be identified by 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 using genetic engineering. VHH antibody domains are easily humanized based on human VH domains that have a highly homologous sequence to that of the VHH antibody domain.

[0062] VHHs can be used as modular building blocks to generate multivalent and / or multispecific antibody constructs, where "multivalent" means that the construct encompasses more than one single domain antibody and "multispecific" means that the construct encompasses a single domain antibody of more than one binding specificity.

[0063] In some cases, the disclosure states that a protein / amino acid sequence A is a "fragment" of another protein / amino acid sequence B. This means that the protein / amino acid sequence A is missing one or more amino acids at the N-terminus and / or one or more amino acids at the C-terminus compared to the protein / amino acid sequence B. Whether a protein / amino acid sequence is missing one or more amino acids at the N-terminus and / or one or more amino acids at the C-terminus compared to another protein / amino acid sequence can be easily determined, for example, when forming a sequence alignment with a program of the BLAST family, for example.

[0064] As will be understood by those skilled in the art, when the present disclosure refers to a "VHH antibody domain or a fragment thereof", the fragment is an antigen-binding fragment. Thus, the fragment binds to the same antigen (i.e., NKp30) as the "full-length" VHH antibody domain according to the present disclosure from which the fragment is derived. Preferably, the fragment of the VHH antibody domain is a C-terminal fragment. This means that the fragment lacks amino acids at the N-terminus compared to the "complete" VHH antibody domain sequence.

[0065] When the present disclosure indicates that a VHH antibody domain or a fragment thereof comprises the complementarity determining regions CDR1, CDR2, and CDR3 "of one of VHH1 to VHH16 as shown in the CDR table", this means that the VHH antibody domain comprises either a combination of CDRs or VHH1 or VHH2 or VHH3 etc., but does not comprise a mixture of CDRs selected from different ones of the listed VHHs. Thus, the VHH antibody domain or fragment thereof includes, by way of example, combinations of CDR1, CDR2 and CDR3 of VHH1 (SEQ ID NOs: 18, 19 and 20), combinations of CDR1, CDR2 and CDR3 of VHH2 (SEQ ID NOs: 21, 22 and 23), or combinations of CDR1, CDR2 and CDR3 of VHH3 (SEQ ID NOs: 24, 25 and 26), etc., but does not include combinations of CDR1 and CDR2 of VHH1 with CDR3 of VHH2 (SEQ ID NOs: 18, 19 and 23).

[0066] When the present disclosure defines a VHH antibody domain or a fragment thereof comprising complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with modifications, where the modifications are, for example, that at least one sequence of CDR1, CDR2 and CDR3 is humanized, the skilled person knows that this humanization exists compared to the corresponding sequence in the CDR table providing the combination of CDR sequences without modifications.

[0067] In the above definition, when the present disclosure indicates the presence of a modification that is "replacement, addition, or deletion" of a number of amino acids (for example, up to 3), the skilled artisan will understand that this is an individual replacement, addition, or deletion. Thus, the replaced, added, or deleted amino acids may be in adjacent positions, or may be in independent, isolated positions in the amino acid sequence. Moreover, as above, the skilled artisan will understand that this definition indicates replacement, addition, or deletion compared to the unmodified sequence in the CDR table.

[0068] In some embodiments, the modification in (b) is that the CDR1 and / or CDR2 sequences are humanized, but not the CDR3 sequence. In some embodiments, the modification in (b) is that the CDR1 sequence is humanized, but not the CDR2 and CDR3 sequences. In some embodiments, the modification in (b) is that the CDR2 sequence, but not the CDR1 and CDR3 sequences, is humanized. In some embodiments, the modification in (b) is that the sequence of one, but not two or more of CDR1, CDR2, and CDR3 is humanized.

[0069] In some embodiments, said humanization of said CDR(s) is by replacing at least one amino acid in the sequence of said CDR with the corresponding amino acid of a human VH domain. In some embodiments, said humanization of said CDR(s) is by replacing up to 3 amino acids in the sequence of said CDR with the corresponding amino acids of a human VH domain. In some embodiments, the humanization of the CDR(s) is by replacing up to 3 amino acids in the CDR1 and / or CDR2 sequence and up to 1 amino acid in the CDR3 sequence with corresponding amino acids from 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 with the corresponding amino acids of a human VH domain. In some embodiments, the humanization of the CDR(s) is by replacing up to 2 amino acids in the CDR1 and / or CDR2 sequence and up to 1 amino acid in the CDR3 sequence with 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 with the corresponding amino acid of a human VH domain.

[0070] In some embodiments, the modification in (c) is - substitutions, additions or deletions of up to 3 amino acids in CDR1, - replacement, addition or deletion of up to 3 amino acids in CDR2, and / or - A maximum of one amino acid replacement, addition or deletion in CDR3.

[0071] In some embodiments, the modification in (c) is - substitution, addition or deletion of up to 2 amino acids in CDR1, - replacement, addition or deletion of up to 2 amino acids in CDR2, and / or - Maximum of 2 amino acid substitutions, additions, or deletions in CDR3 ;

[0072] In some embodiments, the modification in (c) is - replacement, addition or deletion of up to 2 amino acids in CDR1; - a substitution, addition or deletion of up to 2 amino acids in CDR2; and / or - A maximum of one amino acid replacement, addition or deletion in CDR3.

[0073] In some embodiments, the modification in (c) is - replacement, addition or deletion of up to 2 amino acids in CDR1, and / or - a maximum of two amino acid replacements, additions, or deletions in CDR2; Here, the sequence of CDR3 is unmodified.

[0074] As the skilled artisan will understand, indicating that the "CDR3 sequence is unmodified" means that the sequence is unmodified compared to the sequence defining the CDR3 for the VHH in question in the CDR table.

[0075] In some embodiments, the modification in (c) is - a maximum of two amino acid substitutions, additions or deletions in CDR1, Here, the sequences of CDR2 and CDR3 are unmodified.

[0076] In some embodiments, the modification in (c) is - a maximum of two amino acid substitutions, additions or deletions in CDR2, Here, the sequences of CDR1 and CDR3 are unmodified.

[0077] In some embodiments, the modification in (c) is - replacement, addition or deletion of up to one amino acid in CDR1; - a substitution, addition or deletion of up to one amino acid in CDR2; and / or - A maximum of one amino acid replacement, addition or deletion in CDR3.

[0078] In some embodiments, the modification in (c) is - a replacement, addition or deletion of one amino acid in CDR1, and / or - a substitution, addition or deletion of one amino acid in CDR2; Here, the sequence of CDR3 is unmodified.

[0079] In some embodiments, the modification in (c) is - a substitution, addition or deletion of one amino acid in CDR1, Here, the sequences of CDR2 and CDR3 are unmodified.

[0080] In some embodiments, the modification in (c) is - a substitution, addition or deletion of one amino acid in CDR2, Here, the sequences of CDR1 and CDR3 are unmodified.

[0081] In some embodiments, the modifications in (c) include only amino acid replacements, and not additions or deletions of amino acids. In some embodiments, the replacements are conservative amino acid replacements.

[0082] As used herein, "conservative amino acid replacement" refers to the replacement of one amino acid with another, biologically similar amino acid. Conservative replacement is unlikely to change the shape or characteristics of a protein / amino acid sequence. Examples of conservative replacement include the replacement of one hydrophobic residue (such as isoleucine, valine, leucine, or methionine) with another, or the replacement of one polar residue with another (such as the replacement of arginine with lysine, the replacement of glutamic acid with aspartic acid, or the replacement of glutamine with asparagine).

[0083] According to a second aspect, the present disclosure relates to a compound comprising a VHH antibody domain or a fragment thereof, (A) the VHH antibody domain comprises any one of VHH1 to VHH16 shown in the VHH sequence table below; (B) the VHH antibody domain comprises a VHH sequence as defined in (A) with a modification, wherein the modification is that the sequence is humanized; (C) the VHH antibody domain comprises a VHH sequence as defined in (A) with a modification, wherein the modification is a replacement, addition or deletion of up to 25 amino acids; or (D) the VHH antibody domain comprises a VHH sequence that is at least 75% identical to the VHH sequence set forth in (A); Table of VHH sequences: [Table E-1]

[0084] [Table E-2]

[0085] [Table E-3]

[0086] According to a third aspect, the present disclosure relates to a compound comprising a VHH antibody domain or a fragment thereof, (A) the VHH antibody domain consists of any one of VHH1 to VHH16 shown in the VHH sequence table; (B) the VHH antibody domain consists of a VHH sequence as defined in (A) with a modification, wherein the modification is that the sequence is humanized; (C) the VHH antibody domain consists of a VHH sequence as defined in (A) with a modification, wherein the modification is a replacement, addition or deletion of up to 25 amino acids; or (D) The VHH antibody domain consists of a VHH sequence that is at least 75% identical to the VHH sequence mentioned in (A).

[0087] To the second and third aspects the same explanations and definitions provided with respect to the first aspect above apply accordingly. As one of skill in the art will appreciate, when the present disclosure indicates that a VHH antibody domain "comprises any one of the VHH sequences VHH1 to VHH16 shown in the table of VHH sequences," this means that the VHH antibody domain comprises one (and not more than one or all) of the sequences listed in the table of VHH sequences.

[0088] Thus, when the present disclosure indicates that a VHH antibody domain or a fragment thereof comprises / consists of a VHH sequence as defined in (A) with a modification, where the modification is that the sequence is humanized, the skilled person knows that this means that this humanization is present compared to the corresponding sequence in the table of VHH sequences providing the sequence without the modification.

[0089] In the above definition, when the present disclosure indicates the presence of a modification that is a "replacement, addition, or deletion" of a number of amino acids (for example, up to 3), the skilled artisan will understand that this is an individual replacement, addition, or deletion. Thus, the replaced, added, or deleted amino acids may be in adjacent positions or may be in separate, isolated positions within the amino acid sequence. Moreover, as above, the skilled artisan will understand that this definition indicates a replacement, addition, or deletion compared to the unmodified sequence in the table of VHH sequences.

[0090] When the present disclosure states that a sequence A is "at least x% identical" to another sequence B, this is equivalent to the statement that sequence A has "x% identity" with sequence B. The statement reflects the relationship between two polypeptide sequences A and B determined by comparing the sequences. In general, identity refers to an exact amino acid-to-amino acid match of each of the two polypeptide sequences over the length of the sequences being compared. For sequences that are not exactly identical, the percentage that the two sequences are identical may be determined. In general, the two sequences being compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" into either one or both sequences to enhance the degree of alignment. The % identity may be determined over the entire length of each of the sequences being compared (so-called global alignment), which is particularly suitable for sequences of the same or very similar length, or over a shorter defined length (so-called local alignment), which is more suitable for sequences of unequal lengths.

[0091] Methods for comparing the identity of two or more sequences are well known in the art. Thus, for example, the programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux J et al., 1984), such as the programs BESTFIT and GAP, may be used to determine the percent identity between two polynucleotides and the percent identity between two polypeptide sequences. BESTFIT uses the "local homology" algorithm of Smith and Waterman (1981) to find the best single region of similarity between two sequences. Other programs for determining identity sequences are also known in the art, for example the BLAST family of programs (Altschul SF et al, 1990, Altschul SF et al, 1997, accessible through the NCBI homepage 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 SF et al, 1990; Altschul SF et al, 1997, accessible through the NCBI homepage at www.ncbi.nlm.nih.gov).

[0092] In some embodiments, said fragment of said VHH antibody domain comprises at least 75% of the amino acids of said VHH antibody domain sequence. As the skilled artisan will appreciate, this means that the fragment lacks up to a quarter of the total number of amino acids of said VHH antibody domain, where said amino acids are missing at either the N-terminus or C-terminus, compared to the "complete" VHH antibody domain sequence.

[0093] 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, wherein 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.

[0094] In some embodiments, said fragment of said VHH antibody domain comprises the 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 the CDR1 to the C-terminus of the CDR3 of said VHH antibody domain.

[0095] In some embodiments, said fragment of said VHH antibody domain in (A) comprises all the complementarity determining regions (CDRs) of said VHH antibody domain. In some embodiments, said humanization of said sequence in (B) is by replacing at least one amino acid of said sequence with the corresponding amino acid of a human VH (variable heavy) domain. In some embodiments, said humanization of said sequence in (B) is by replacing up to 25 amino acids of said sequence (individually) by the corresponding amino acids of a human VH domain.

[0096] As will be understood by those of skill in the art, when the present disclosure refers to the replacement of an amino acid in a sequence / domain A with a "corresponding" amino acid in a sequence / domain B, this specifies that said amino acid in sequence / domain A is replaced by an amino acid in sequence / domain B that aligns with said amino acid in sequence / domain A in an alignment of the two sequences.

[0097] In some embodiments, said humanization of said sequence in (B) is by replacing up to 20 amino acids of said sequence by the corresponding amino acids of a human VH domain. In some embodiments, said humanization of said sequence in (B) is by replacing up to 15 amino acids of said sequence by corresponding amino acids of a human VH domain. In some embodiments, said humanization of said sequence in (B) is by replacing up to 10 amino acids of said sequence by the corresponding amino acids of a human VH domain. In some embodiments, said humanization of said sequence in (B) is by replacing up to 5 amino acids of said sequence by the corresponding amino acids of a human VH domain. In some embodiments, said humanization of said sequence in (B) is by replacing up to 3 amino acids of said sequence by the corresponding amino acids of a human VH domain. In some embodiments, said humanization of said sequence in (B) is by replacing up to 2 amino acids of said sequence by the corresponding amino acids of a human VH domain. In some embodiments, said humanization of said sequence in (B) is by replacing one amino acid of said sequence with the corresponding amino acid of a human VH domain.

[0098] In some embodiments, the humanization in (B) is within the framework regions of said VHH antibody domain and / or within the CDRs of said VHH antibody domain. In some embodiments, the humanization in (B) is within the framework regions of said VHH antibody domain but not within the CDRs of said VHH antibody domain. In some embodiments, said humanization in (B) is within the CDRs of said VHH antibody domain but not within the framework regions of said VHH antibody domain.

[0099] In some embodiments, said humanization within the CDRs of said VHH antibody domain in (B) is within CDR1, CDR2, and / or CDR3. In some embodiments, said humanization within the CDRs of said VHH antibody domain in (B) is within CDR1 and / or CDR2. In some embodiments, said humanization within a CDR of said VHH antibody domain in (B) is within CDR1. In some embodiments, said humanization within a CDR of said VHH antibody domain in (B) is within CDR2. In some embodiments, said humanization within a CDR of said VHH antibody domain in (B) is not within CDR3.

[0100] In some embodiments, the modifications in (C) are substitutions, additions, or deletions of up to 20 amino acids. In some embodiments, the modifications in (C) are substitutions, additions, or deletions of up to 15 amino acids. In some embodiments, the modifications in (C) are substitutions, additions, or deletions of up to 10 amino acids. In some embodiments, the modification in (C) is a substitution, addition, or deletion of up to 5 amino acids. In some embodiments, the modification in (C) is a substitution, addition, or deletion of up to 3 amino acids. In some embodiments, the modification in (C) is a substitution, addition, or deletion of up to two amino acids. In some embodiments, the modification in (C) is a single amino acid replacement, addition, or deletion. In some embodiments, the modifications in (c) include only amino acid replacements, and not amino acid additions or deletions.

[0101] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises the complementarity determining regions CDR1, CDR2, and CDR3 of one of VHH1 to VHH16 as shown in the CDR table.

[0102] 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 of VHH1 to VHH16 as shown in the CDR Table; (b) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with a modification, wherein the modification is that at least one sequence of CDR1, CDR2 and CDR3 is humanized; (c) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with a modification, wherein the modification is - substitutions, additions or deletions of up to 3 amino acids in CDR1, - replacement, addition or deletion of up to 3 amino acids in CDR2, and / or - Substitution, addition or deletion of up to 3 amino acids in the CDR3.

[0103] In some embodiments, the modification in (b) is that the CDR1 and / or CDR2 sequences are humanized, but not the CDR3 sequence. In some embodiments, the modification in (b) is that the CDR1 sequence is humanized, but not the CDR2 and CDR3 sequences. In some embodiments, the modification in (b) is that the CDR2 sequence, but not the CDR1 and CDR3 sequences, is humanized. In some embodiments, the modification in (b) is that the sequence of one, but not two or more of CDR1, CDR2, and CDR3 is humanized.

[0104] In some embodiments, said humanization of said CDR(s) is by replacing at least one amino acid in the sequence of said CDR with the corresponding amino acid of a human VH domain. In some embodiments, said humanization of said CDR(s) is by replacing up to 3 amino acids in the sequence of said CDR with the corresponding amino acids of a human VH domain. In some embodiments, the humanization of the CDR(s) is by replacing up to 3 amino acids in the CDR1 and / or CDR2 sequence and up to 1 amino acid in the CDR3 sequence with corresponding amino acids from 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 with the corresponding amino acids of a human VH domain. In some embodiments, the humanization of the CDR(s) is by replacing up to 2 amino acids in the CDR1 and / or CDR2 sequence and up to 1 amino acid in the CDR3 sequence with 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 with the corresponding amino acid of a human VH domain.

[0105] In some embodiments, the modification in (c) is - substitutions, additions or deletions of up to 3 amino acids in CDR1, - replacement, addition or deletion of up to 3 amino acids in CDR2, and / or - A maximum of 2 amino acid replacements, additions, or deletions in the CDR3.

[0106] In some embodiments, the modification in (c) is - substitutions, additions or deletions of up to 3 amino acids in CDR1, - replacement, addition or deletion of up to 3 amino acids in CDR2, and / or - A maximum of one amino acid replacement, addition or deletion in CDR3.

[0107] In some embodiments, the modification in (c) is - substitutions, additions or deletions of up to 3 amino acids in CDR1, and / or - a substitution, addition or deletion of up to 3 amino acids in CDR2, Here, the sequence of CDR3 is unmodified. As the skilled artisan will appreciate, "unmodified" means unmodified compared to the sequence in the CDR table.

[0108] In some embodiments, the modification in (c) is - substitution, addition or deletion of up to 2 amino acids in CDR1, - replacement, addition or deletion of up to 2 amino acids in CDR2, and / or - replacement, addition or deletion of up to 2 amino acids in CDR3;

[0109] In some embodiments, the modification in (c) is - replacement, addition or deletion of up to 2 amino acids in CDR1; - a substitution, addition or deletion of up to 2 amino acids in CDR2; and / or - A maximum of one amino acid replacement, addition or deletion in CDR3.

[0110] In some embodiments, the modification in (c) is - replacement, addition or deletion of up to 2 amino acids in CDR1, and / or - a maximum of two amino acid replacements, additions, or deletions in CDR2; Here, the sequence of CDR3 is unmodified.

[0111] In some embodiments, the modification in (c) is - a maximum of two amino acid substitutions, additions or deletions in CDR1, Here, the sequences of CDR2 and CDR3 are unmodified.

[0112] In some embodiments, the modification in (c) is - a maximum of two amino acid substitutions, additions or deletions in CDR2, Here, the sequences of CDR1 and CDR3 are unmodified.

[0113] In some embodiments, the modification in (c) is - replacement, addition or deletion of up to one amino acid in CDR1; - a substitution, addition or deletion of up to one amino acid in CDR2; and / or - A maximum of one amino acid replacement, addition or deletion in CDR3.

[0114] In some embodiments, the modification in (c) is - a replacement, addition or deletion of one amino acid in CDR1, and / or - a substitution, addition or deletion of one amino acid in CDR2; Here, the sequence of CDR3 is unmodified.

[0115] In some embodiments, the modification in (c) is - a substitution, addition or deletion of one amino acid in CDR1, Here, the sequences of CDR2 and CDR3 are unmodified.

[0116] In some embodiments, the modification in (c) is - a substitution, addition or deletion of one amino acid in CDR2, Here, the sequences of CDR1 and CDR3 are unmodified.

[0117] In some embodiments, the modifications in (c) include only amino acid replacements, and not amino acid additions or deletions.

[0118] In some embodiments, the compound comprises a VHH antibody domain (including but not limited to a fragment thereof). In some embodiments, the compound consists of a fragment of a VHH antibody domain (not a full-length VHH antibody domain). In some embodiments, the fragment consists of at least 100 amino acids. In some embodiments, the fragment consists of at least 105 amino acids. In some embodiments, the fragment consists of at least 110 amino acids. In some embodiments, the fragment consists of at least 115 amino acids.

[0119] In some embodiments, the compound is capable of specifically binding to NKp30. In some embodiments, the specific term that a compound, domain, or fragment is "capable of specifically binding to NKp30" means that the compound, domain, or fragment is capable of binding to NKp30 with an affinity at least equal to that with which human B7-H6 (SEQ ID NO: 17) binds to NKp30. Such binding can be determined by in vitro binding experiments (by biolayer interference) as described in Example 1 below.

[0120] In some embodiments, the VHH antibody domain or fragment thereof is capable of specifically binding to NKp30. In some embodiments, the VHH antibody domain or fragment thereof is administered to recombinant human NKp30 at 1x10-6 Binds at or stronger than the KD of M. In some embodiments, the VHH antibody domain or fragment thereof is administered to recombinant human NKp30 at 1x10 -7 Binds at or stronger than the KD of M. In some embodiments, the VHH antibody domain or fragment thereof is administered to recombinant human NKp30 at 1x10 -8 Binds at or stronger than the KD of M. In some embodiments, the VHH antibody domain or fragment thereof is administered to recombinant human NKp30 at 1x10 -9 Binds at or stronger than the KD of M.

[0121] In some embodiments, the VHH antibody domain or fragment thereof is administered to recombinant human NKp30 at 1x10 4 k greater than or equal to (1 / Ms) on Combine with. In some embodiments, the VHH antibody domain or fragment thereof is administered to recombinant human NKp30 at 1x10 5 k greater than or equal to (1 / Ms) on Combine with.

[0122] In some embodiments, the VHH antibody domain or fragment thereof is administered to recombinant human NKp30 in a 5x10 -3 k less than (1 / Ms) off Combine with. In some embodiments, the VHH antibody domain or fragment thereof is administered to recombinant human NKp30 at 1x10 -3 k less than (1 / Ms) off Combine with. In some embodiments, the VHH antibody domain or fragment thereof is administered to recombinant human NKp30 at 1x10 -4 k less than (1 / Ms) off Combine with. In some embodiments, the VHH antibody domain or fragment thereof is administered to recombinant human NKp30 at 1x10 -5 k less than (1 / Ms) off Combine with. In some embodiments, the VHH antibody domain or fragment thereof is administered to recombinant human NKp30 at 1x10 -6 k less than (1 / Ms) off Combine with. In some embodiments, the VHH antibody domain or fragment thereof is administered to recombinant human NKp30 at 1x10 -7 k less than (1 / Ms) off Combine with.

[0123] In some embodiments, the VHH antibody domain or fragment thereof in (b) and (c) binds to recombinant human NKp30 with an affinity (KD value) that is no more than 5-fold weaker than the binding of the corresponding VHH antibody domain without the modification. In some embodiments, the VHH antibody domain or fragment thereof in (b) and (c) binds to recombinant human NKp30 with an affinity (KD value) that is no more than 2-fold weaker than the binding of the corresponding VHH antibody domain without the modification. In some embodiments, the VHH antibody domain or fragment thereof in (b) and (c) binds to recombinant human NKp30 with an affinity (KD value) that is no more than 1.5-fold weaker than the binding of the corresponding VHH antibody domain without the modification.

[0124] In some embodiments, the VHH antibody domain or fragment thereof in (b) and (c) binds to recombinant human NKp30 with an affinity (KD value) that is not more than 5-fold stronger than the binding of the corresponding VHH antibody domain without the modification. In some embodiments, the VHH antibody domain or fragment thereof in (b) and (c) binds to recombinant human NKp30 with an affinity (KD value) that is not more than 2-fold stronger than the binding of the corresponding VHH antibody domain without the modification. In some embodiments, the VHH antibody domain or fragment thereof in (b) and (c) binds to recombinant human NKp30 with an affinity (KD value) that is not more than 1.5-fold stronger than the binding of the corresponding VHH antibody domain without the modification.

[0125] In some embodiments, the VHH antibody domains in (B) and (C) bind to recombinant human NKp30 with an affinity (KD value) that is no more than 5-fold weaker than the binding of the corresponding VHH antibody domain without modification. In some embodiments, the VHH antibody domains in (B) and (C) bind to recombinant human NKp30 with an affinity (KD value) that is no more than 2-fold weaker than the binding of the corresponding VHH antibody domain without the modification. In some embodiments, the VHH antibody domains in (B) and (C) bind to recombinant human NKp30 with an affinity (KD value) that is no more than 1.5-fold weaker than the binding of the corresponding VHH antibody domain without modification.

[0126] In some embodiments, the VHH antibody domains in (B) and (C) bind to recombinant human NKp30 with an affinity (KD value) that is no more than 5-fold stronger than the binding of the corresponding VHH antibody domain without modification. In some embodiments, the VHH antibody domains in (B) and (C) bind to recombinant human NKp30 with an affinity (KD value) that is not more than 2-fold stronger than the binding of the corresponding VHH antibody domain without modification. In some embodiments, the VHH antibody domains in (B) and (C) bind to recombinant human NKp30 with an affinity (KD value) that is not more than 1.5-fold stronger than the binding of the corresponding VHH antibody domain without modification.

[0127] In some embodiments, the affinity of binding (KD value) of the VHH antibody domain in (D) to human NKp30 is not more than 5-fold weaker than the affinity of binding (KD value) of a VHH antibody domain consisting of a sequence from the table of VHH sequences having the highest sequence identity with the sequence of the VHH antibody domain in (D). The degree of sequence identity may be determined by sequence alignment. In some embodiments, the affinity of binding (KD value) of the VHH antibody domain in (D) to human NKp30 is not more than two-fold weaker than the affinity of binding (KD value) of a VHH antibody domain consisting of a sequence from the table of VHH sequences having the highest sequence identity to the sequence of the VHH antibody domain in (D) to human NKp30. In some embodiments, the affinity of binding (KD value) of the VHH antibody domain in (D) to human NKp30 is not more than 1.5 times weaker than the affinity of binding (KD value) of a VHH antibody domain consisting of a sequence from the table of VHH sequences having the highest sequence identity to the sequence of the VHH antibody domain in (D) to human NKp30.

[0128] In some embodiments, the binding affinity (KD value) of the VHH antibody domain in (D) to human NKp30 is not more than 5 times stronger than the binding affinity (KD value) of a VHH antibody domain consisting of a sequence from the table of VHH sequences having the highest sequence identity to the sequence of the VHH antibody domain in (D) to human NKp30. In some embodiments, the affinity of binding (KD value) of the VHH antibody domain in (D) to human NKp30 is not more than two-fold stronger than the affinity of binding (KD value) of a VHH antibody domain consisting of a sequence from the table of VHH sequences having the highest sequence identity to the sequence of the VHH antibody domain in (D) to human NKp30. In some embodiments, the binding affinity (KD value) of the VHH antibody domain in (D) to human NKp30 is not more than 1.5 times stronger than the binding affinity (KD value) of a VHH antibody domain consisting of a sequence from the table of VHH sequences having the highest sequence identity to the sequence of the VHH antibody domain in (D) to human NKp30.

[0129] In some embodiments, the KD value / the k on speed / k off The rate is measured by kinetic measurement by biolayer interference in KB buffer (PBS + 0.1% Tween-20 + 1% BSA) at 25°C and 1000 rpm. In some embodiments, the binding (i.e., the binding / specific binding itself) is determined by this approach.

[0130] In some embodiments, the VHH antibody domain or fragment thereof competes with human B7-H6 for binding to human NKp30. If the VHH antibody domain or fragment thereof competes with human B7-H6, this can be determined as described in the Examples section.

[0131] In some embodiments, the VHH antibody domain or fragment thereof does not compete with human B7-H6 for binding to human NKp30. In some embodiments, the VHH antibody domain or fragment thereof partially competes with human B7-H6 for binding to human NKp30.

[0132] In some embodiments, the VHH antibody domain of (A) comprises any one of the VHH sequences VHH1, VHH2, VHH3, VHH4, VHH5, VHH8, VHH9, VHH10, VHH11, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the Table of VHH Sequences. (For compounds having such VHH antibody domains, a KD in the range of E-09 or stronger was observed for binding to NKp30, as can be seen, for example, from Example 3, Table 1.)

[0133] In some embodiments, the VHH antibody domain of (A) comprises any one of the VHH sequences VHH1, VHH3, VHH5, VHH8, VHH10, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the Table of VHH Sequences. (For compounds having such VHH antibody domains, a KD in the range of E-10 or stronger was observed for binding to NKp30, as can be seen, for example, from Example 3, Table 1.)

[0134] In some embodiments, the VHH antibody domain of (A) comprises the VHH sequence VHH15 as shown in the table of VHH sequences. (For compounds having such a VHH antibody domain, as can be seen, for example, from Example 3, Table 1, a KD in the range of E-12 or stronger and a kD in the range of E-07 or weaker.) off was observed for binding to NKp30.

[0135] In some embodiments, the VHH antibody domain of (A) comprises any one of the VHH sequences VHH1, VHH2, VHH3, VHH5, VHH6, VHH8, VHH9, VHH10, VHH11, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the Table of VHH Sequences. (For compounds having such VHH antibody domains, k in the range of E-05, as can be seen from, for example, Example 3, Table 1.) on was observed for binding to NKp30.

[0136] In some embodiments, the VHH antibody domain of (A) comprises any one of the VHH sequences VHH1, VHH2, VHH3, VHH4, VHH5, VHH8, VHH10, VHH11, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the Table of VHH Sequences. (For compounds having such VHH antibody domains, as can be seen, for example, in Example 3, Table 1, k in the range of E-04 or lower. off was observed for binding to NKp30.

[0137] In some embodiments, the VHH antibody domain of (A) comprises any one of the VHH sequences VHH10, VHH15, or VHH16 shown in the Table of VHH Sequences. (For compounds having such VHH antibody domains, as can be seen, for example, in Example 3, Table 1, k in the range of E-05 or lower. off was observed for binding to NKp30.

[0138] In some embodiments, the VHH antibody domain of (A) comprises any one of the VHH sequences VHH1, VHH3, VHH5, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the table of VHH sequences. (For compounds having such VHH antibody domains, competition with B7-H6 is observed, as can be seen, for example, from Example 3, Table 1. As shown in Example 4, such VHH antibody domains result in compounds that mediate lysis of targeted cells with high efficiency.)

[0139] In some embodiments, the VHH antibody domain of (A) comprises any one of the VHH sequences VHH1, VHH5, or VHH16 as shown in the Table of VHH Sequences.

[0140] In some embodiments, the VHH antibody domain of (A) comprises any one of the VHH sequences VHH2, VHH6, VHH7, VHH8, VHH9, VHH10, or VHH11 shown in the table of VHH sequences. (For compounds with such VHH antibody domains, no competition with B7-H6 is observed, as can be seen, for example, from Example 3, Table 1. As shown in Example 4, such VHH antibody domains result in compounds that mediate lysis of targeted cells, but with less efficiency.)

[0141] In some embodiments, the VHH antibody domain of (A) comprises any one of the VHH sequences VHH2, VHH4, or VHH8 as shown in the Table of VHH Sequences. In some embodiments, the VHH antibody domain of (A) comprises any one of the VHH sequences VHH2, or VHH8 shown in the Table of VHH Sequences.

[0142] In some embodiments, the VHH antibody domain of (A) comprises the VHH sequence VHH4 shown in the Table of VHH Sequences. (For compounds having such a VHH antibody domain, partial competition with B7-H6 is observed, as can be seen, for example, from Example 3, Table 1.)

[0143] In some embodiments, the VHH antibody domain of (A) comprises any one of the VHH sequences VHH12 or VHH15 shown in the Table of VHH Sequences. (For compounds with such VHH antibody domains, no competition is observed with the partial competitor VHH4, as can be seen, for example, from Example 3.)

[0144] In some embodiments, the VHH antibody domain of (A) comprises any one of the VHH sequences VHH8, VHH9, VHH10, or VHH11 shown in the Table of VHH Sequences. (As can be seen, for example, in Example 3, compounds with such VHH antibody domains belong to bins of specific epitopes that share common characteristics.)

[0145] In some embodiments, the VHH antibody domain of (A) comprises any one of the VHH sequences VHH2 or VHH6 shown in the table of VHH sequences. (As can be seen, for example, in Example 3, compounds with such VHH antibody domains belong to bins of specific epitopes with common features.)

[0146] In some embodiments, the VHH antibody domain of (A) comprises any one of the VHH sequences VHH2, VHH6, or VHH7 shown in the Table of VHH Sequences. (As can be seen, for example, in Example 3, compounds with such VHH antibody domains are unique with respect to their targeting of epitopes.)

[0147] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of any one of the VHH sequences VHH1, VHH2, VHH3, VHH4, VHH5, VHH8, VHH9, VHH10, VHH11, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the CDR table. (For compounds having such VHH antibody domains, a KD in the range of E-09 or stronger was observed for binding to NKp30, as can be seen, for example, from Example 3, Table 1.)

[0148] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of any one of VHH1, VHH3, VHH5, VHH8, VHH10, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the CDR table. (For compounds having such VHH antibody domains, a KD in the range of E-10 or stronger was observed for binding to NKp30, as can be seen, for example, from Example 3, Table 1.)

[0149] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH15 as shown in the CDR table. (For a compound having such a VHH antibody domain, as can be seen, for example, in Example 3, Table 1, it has a KD in the range of E-12 or higher and a kD in the range of E-07 or lower. off was observed for binding to NKp30.

[0150] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH1, VHH2, VHH3, VHH5, VHH6, VHH8, VHH9, VHH10, VHH11, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the CDR table. (For compounds having such VHH antibody domains, for example, k in the range of E-05, as can be seen from Example 3, Table 1.) on was observed for binding to NKp30.

[0151] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH1, VHH2, VHH3, VHH4, VHH5, VHH8, VHH10, VHH11, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the CDR table. (For compounds having such VHH antibody domains, k values ​​in the range of E-04 or lower, as can be seen, for example, from Example 3, Table 1.) off was observed for binding to NKp30.

[0152] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH10, VHH15, or VHH16 as shown in the CDR table. (For compounds having such VHH antibody domains, k values ​​in the range of E-05 or lower, as can be seen, for example, from Example 3, Table 1.) off was observed for binding to NKp30.

[0153] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH1, VHH3, VHH5, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the CDR table. (For compounds having such VHH antibody domains, as can be seen, for example, from Example 3, Table 1, competition with B7-H6 is observed. As shown in Example 4, such VHH antibody domains result in compounds that mediate lysis of targeted cells with high efficiency.)

[0154] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH1, VHH5, or VHH16 as shown in the CDR Table.

[0155] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH2, VHH6, VHH7, VHH8, VHH9, VHH10, or VHH11 as shown in the CDR table. (For compounds with such VHH antibody domains, no competition with B7-H6 is observed, as can be seen, for example, from Example 3, Table 1. As shown in Example 4, such VHH antibody domains result in compounds that mediate lysis of targeted cells, but with less efficiency.)

[0156] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH2, VHH4, or VHH8 as shown in the CDR table. In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH2 or VHH8 as shown in the CDR table.

[0157] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises the complementarity determining regions CDR1, CDR2, and CDR3 of VHH4 as shown in the CDR table. (For compounds having such VHH antibody domains, partial competition with B7-H6 is observed, as can be seen, for example, from Example 3, Table 1.)

[0158] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH12 or VHH15 as shown in the CDR table. (For compounds with such VHH antibody domains, no competition is observed with the partial competitor VHH4, as can be seen, for example, from Example 3.)

[0159] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH8, VHH9, VHH10, or VHH11 as shown in the CDR table. (As can be seen, for example, in Example 3, compounds with such VHH antibody domains belong to bins of specific epitopes with common features.)

[0160] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH2 or VHH6 shown in the CDR table. (As can be seen, for example, in Example 3, compounds with such VHH antibody domains belong to bins of specific epitopes with common features.)

[0161] In some embodiments, the VHH antibody domain or fragment thereof in (A) comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH2, VHH6, or VHH7 shown in the CDR table. (As can be seen, for example, from Example 3, compounds with such VHH antibody domains are unique with respect to their targeting of epitopes.)

[0162] In some embodiments, the compound is a molecule. In some embodiments, the compound comprises or is a protein.

[0163] By stating that a compound "comprises" a protein, the present disclosure specifies that the compound encompasses within its chemical structure moieties that are proteins. A compound that includes a protein may or may not also include non-protein moieties. In some embodiments, the compound is a protein.

[0164] By stating that a compound "is" a protein, the present disclosure specifies that the compound consists solely of proteins, and does not include non-proteinaceous moieties. In some embodiments, the VHH antibody domain is derived from a camelid antibody, hi some embodiments, the VHH antibody domain is derived from a llama antibody.

[0165] In some embodiments, the compound comprises a VHH antibody domain or a fragment thereof according to (A), (B), or (C). In some embodiments, the compound comprises a VHH antibody domain or a fragment thereof according to (A), (B), or (D). In some embodiments, the compound comprises a VHH antibody domain or a fragment thereof according to (A), (C), or (D). In some embodiments, the compound comprises a VHH antibody domain or a fragment thereof according to (A) or (B). In some embodiments, the compound comprises a VHH antibody domain or a fragment thereof according to (A) or (C). In some embodiments, the compound comprises a VHH antibody domain or a fragment thereof according to (A) or (D). In some embodiments, the compound comprises a VHH antibody domain or a fragment thereof according to (A). In some embodiments, the compound comprises a VHH antibody domain or a fragment thereof according to (B).

[0166] In some embodiments, said VHH antibody domain or fragment thereof comprises a complementarity determining region according to (a) or (b). In some embodiments, said VHH antibody domain or fragment thereof comprises a complementarity determining region according to (a) or (c). In some embodiments said VHH antibody domain or fragment thereof comprises a complementarity determining region according to (a). In some embodiments, said VHH antibody domain or fragment thereof comprises a complementarity determining region according to (b).

[0167] In some embodiments, the 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 associates (typically covalently) with the compound and binds to a target site, where the binding allows the compound to be recruited to the target site. A target site will typically be a biological molecule or a portion of a biological molecule. An example of a targeting moiety is an antigen-binding antibody fragment that is covalently linked to a NKp30-binding VHH antibody domain to form a compound according to the present disclosure, where the antigen-binding fragment binds to a receptor (the antigen) present on the surface of a cell type, and where the binding of the antigen-binding fragment to the receptor results in the recruitment of the compound to the cell type.

[0168] Non-targeted drugs typically reach their sites of action through systemic distribution and passive diffusion. In contrast, targeted compounds are not distributed evenly throughout the body. Due to the interaction of the targeting moiety with its target molecule, compounds that include a targeting moiety are preferentially concentrated at their target site. Thus, for example, therapeutic compounds with a targeting moiety require a smaller dosage to be therapeutically effective, thus improving the therapeutic window.

[0169] In some embodiments, all components of the compound are covalently linked. In some embodiments, the targeting moiety is a group of molecules that specifically binds to a target molecule or a fragment thereof. In some embodiments, the target molecule is a receptor on the surface of a cell. In some embodiments, the target molecule is an antigen present on the surface of a target cell.

[0170] As used herein, a "target molecule present on the surface of a target cell" is a molecule present on the surface of a target cell in such a way that it is accessible from the extracellular environment (i.e., an antibody can bind to it from the extracellular environment, for example). For example, CD8 is a transmembrane protein of cytotoxic T cells, the extracellular domain of which is accessible from the extracellular environment to an antibody directed against the extracellular domain of CD8. Thus, in the sense of this disclosure, CD8 is a target molecule present on the surface of a cytotoxic T cell.

[0171] A targeting moiety that "binds" to a target molecule of interest is one that is capable of binding to that target molecule with sufficient affinity such that the targeting moiety is useful for targeting a compound to a cell expressing the target molecule.

[0172] When the present disclosure refers to a first molecule / group of molecules (e.g. an antibody / antibody component) "binding" / "specifically binding" to a second molecule / group of molecules (e.g. an antigen of interest), this means that the first molecule / group of molecules (in this example an antibody) binds to the second molecule / group of molecules (in this example the antigen of interest) with an affinity that is at least 10 times greater than its affinity to other molecules / groups of molecules, in particular to other molecules / groups of molecules in the human body (in this example, its affinity is at least 10 times greater than its binding to non-specific antigens (e.g. BSA, casein) other than the antigen of interest (or closely related antigens). In a preferred embodiment, a first molecule / group of molecules (e.g., an antibody / antibody component) that "specifically binds" to a second molecule / group of molecules (e.g., an antigen of interest) binds to the antigen with an affinity that is at least 100 times greater than its affinity to other molecules / groups of molecules, particularly other molecules / groups in the human body (in this example, its affinity is at least 100 times greater than its binding to non-specific antigens other than the antigen of interest (or closely related antigens). Typically, the binding will be determined under physiological conditions. A first molecule / group of molecules that "specifically binds" to a second molecule / group of molecules will bind to that second molecule / group with an affinity of at least about 1×10 7 M -1 may bind with an affinity of

[0173] In some embodiments, the targeting moiety is a protein, peptide, peptidomimetic, nucleic acid, oligonucleotide, or a small molecule. As used herein, the term "peptidomimetic" refers to a peptide-like chain designed to mimic a peptide. An example of a peptidomimetic is, but is not limited to, a D-peptide mimetic that contains D-amino acids.

[0174] As used herein, a "small molecule" is a molecule with a molecular weight <1000 Da. In some embodiments, the targeting moiety comprises or is a protein. In some embodiments, the targeting moiety comprises a protein. In some embodiments, the targeting moiety is a protein. In some embodiments, the targeting moiety is a protein ligand that specifically binds to a receptor on the surface of a cell. In some embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof. In some embodiments, the targeting moiety is capable of specifically binding to a tumor-associated antigen.

[0175] In some embodiments, the compound is a bispecific antibody. When the present disclosure states that a compound is a bispecific antibody, it does not exclude the possibility that the bispecific antibody is linked to additional domains or moieties. In some embodiments, the compounds are bispecific antibodies prepared by SEED (strand-exchange engineered domain) technology. The term "bispecific antibody" as used in this disclosure refers to an antibody that is capable of specifically binding to two different epitopes simultaneously.

[0176] The terms "epitope" or "antigenic determinant" are used interchangeably herein and refer to a portion of an antigen that is recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed from both contiguous and non-contiguous amino acids arranged side-by-side by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturation, whereas epitopes formed by tertiary folding are typically lost upon protein denaturation. Epitopes typically encompass at least 3 or more, often at least 5 or 8-10 amino acids in a unique spatial conformation.

[0177] The two different epitopes that bispecific antibodies bind to 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 co-expression of two immunoglobulin heavy / light chain pairs (see for example Milstein et al., Nature (1983), vol. 305, p. 537-539). Alternatively, bispecific antibodies can be prepared using chemical linkage (see for example Brennan et al., Science (1985), vol. 229, p. 81). Bispecific antibodies can also be prepared, for example, by SEED technology (an approach for the generation of bispecific antibodies in which structurally related sequences in 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.

[0178] In some embodiments, one binding site of said bispecific antibody is formed by said VHH antibody domain or a fragment thereof and one binding site of said bispecific antibody is formed by said targeting moiety. In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a Fab', a (Fab')2, an Fv, a scFv, a diabody, and a VHH.

[0179] "Fab" fragments are obtained by papain digestion of antibodies, which produces two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment (a designation reflecting the ease with which it can be crystallized). The Fab fragment consists of an entire L chain together with the variable region domain of the H chain (VH) and the first constant region of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.

[0180] "F(ab')2" fragments can be obtained by pepsin treatment of an antibody, which produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with distinct antigen-binding activities and are still capable of cross-linking antigen.

[0181] "Fab'" fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH designates Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0182] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of the antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain types of cells.

[0183] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight non-covalent association. The folding of these two domains results in six highly variable loops (three loops each from the H and L chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or even a half Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0184] A "single-chain Fv", also abbreviated as "scFv", is an antibody fragment comprising a single polypeptide chain comprising a VH antibody domain and a VL antibody domain connected thereto. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun, in: The Pharmacology of Monoclonal Antibodies, vol. 113 (1994), editors Rosenburg and Moore, Springer-Verlag (New York), p. 269-315.

[0185] The term "diabody" refers to small antibody fragments prepared by constructing scFv fragments (see previous paragraph) with a short linker (about 5-25 residues) between the VH and VL domains, such that interchain (but not intrachain) pairing of the V domains is achieved, thereby resulting in a bivalent fragment (i.e., a fragment with 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 more detail, for example, in EP 0404097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA (1993), vol. 90, p. 6444-6448.

[0186] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, and Fv. In some embodiments, the antigen-binding fragment is a Fab. In some embodiments, the antigen-binding fragment is selected from the group consisting of an scFv, a diabody, and a VHH. In some embodiments, the antigen-binding fragment is an antigen-binding fragment of an antibody having a SEED (strand exchange engineered domain) format.

[0187] In some embodiments, the targeting moiety is capable of specifically binding to an antigen present on the surface of a target cell. In some embodiments, the antibody is directed to an antigen present on the surface of a target cell. In some embodiments, the antigen-binding fragment is an antigen-binding fragment of an antibody directed to an antigen present on the surface of a target cell.

[0188] An antibody / antigen-binding fragment "against" an antigen is an antibody / antigen-binding fragment that has an antigen-binding site that binds to that antigen. Whether an antibody / antigen-binding fragment binds to an antigen can be determined, for example, by testing whether the antibody binds to cells expressing the antigen on their cell surface in cultured cells in an immunofluorescence experiment.

[0189] In some embodiments, the antigen present on the surface of the target cell is more abundant on the surface of the target cell than on the surface of other cell types.

[0190] The abundance of a surface antigen on a cell type can be determined by standard methods known to those of skill in the art, such as by flow cytometry (e.g., by exposing cells of the cell type to an antibody of interest, followed by staining with a fluorescently labeled secondary antibody directed against the antibody of interest, and detecting the fluorescent label by flow cytometry).

[0191] In some embodiments, the antigen present on the surface of the target cell is present on the surface of the target cell, but is substantially absent on the surface of other cell types.

[0192] As used herein, an antigen that is "present on the surface of the target cell, but substantially absent from the surface of other cell types" is sufficiently abundant on the surface of the target cell to permit recruitment of a compound bearing a targeting moiety (an antibody or antigen-binding fragment thereof) to the antigen under physiological conditions. In contrast, the abundance of the antigen on the surface of other cell types is low enough that recruitment of the compound under physiological conditions barely exceeds background binding.

[0193] In some embodiments, the antigen present on the surface of the target cell is present on the surface of the target cell but not on the surface of other cell types.

[0194] As used herein, an antigen that is "present on the surface of the target cell, but not on the surface of other cell types" is sufficiently abundant on the surface of the target cell to allow recruitment of a compound bearing a targeting moiety (an antibody or antigen-binding fragment thereof) to the antigen under physiological conditions. In contrast, the abundance of the antigen on the surface of other cell types is low enough that recruitment of the compound under physiological conditions does not exceed background binding.

[0195] In some embodiments, the binding of the targeting moiety to the antigen present on the surface of the target cell can specifically recruit a compound to the target cell.

[0196] The term "antibody-drug conjugate capable of specifically recruiting to the target cells" means that the compound is recruited to the target cells under physiological conditions with an efficiency that is at least 10-fold greater, and preferably at least 100-fold greater, than to other cell types (i.e., to other cell types to which the compound may be exposed in the body during administration of the compound).

[0197] In some embodiments, the antigen present on the surface of the target cell is a tumor-associated antigen.

[0198] As used herein, a "tumor-associated antigen" in its broadest sense is an antigen that allows recruitment of an ADC to a tumor site so that a therapeutic effect or diagnosis (e.g., labeling of the tumor site) can be achieved. A tumor-associated antigen may be either an antigen present on the surface of a tumor cell or an antigen associated with the tumor microenvironment.

[0199] Sources of information on how to identify and validate cell surface expression and tumor-associated antigens are known to the skilled artisan and described in the literature (see, for example, Bornstein, 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: / / oasis-genomics.org / )).

[0200] In preferred embodiments, the tumor-associated antigen is an antigen present on the surface of tumor cells. In these embodiments, the term "tumor-associated antigen" refers to an antigen present on the cell surface of tumor cells and capable of distinguishing tumor cells from other cell types. A tumor-associated antigen may be a part of a molecule (e.g., a protein) that is expressed by tumor cells and accessible from the extracellular environment. A tumor-associated antigen may also be different (i.e., qualitatively different) from its counterpart in a corresponding non-tumor cell (e.g., where the molecule is a protein by one or more amino acid residues). Alternatively, a tumor-associated antigen may be identical to its counterpart in a corresponding non-tumor cell, but present on the surface of tumor cells at a higher level than on the surface of the corresponding non-tumor cell. For example, a tumor-associated antigen may be present only on the surface of tumor cells, but not on the surface of non-tumor cells, or a 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 some embodiments, a tumor-associated antigen is present on the surface of tumor cells at a level at least 1000-fold higher than on the surface of non-tumor cells.

[0201] In some embodiments, the targeting moiety is capable of specifically binding to a tumor-associated antigen. In some embodiments, the tumor-associated antigen is an antigen present on the surface of a tumor cell. In some embodiments, the tumor-associated antigen is EGFR (epidermal growth factor receptor).

[0202] In some embodiments, the compound is a bispecific or multispecific molecule. In some embodiments, the compound is a bispecific molecule. In some embodiments, the compound is a multispecific molecule. In some embodiments, the compound is a bispecific or multispecific molecule that binds to NKp30 via its VHH antibody domain or a fragment thereof and binds to tumor-associated antigen via its targeting domain. In some embodiments, the compound comprises an antibody Fc region.

[0203] 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 the heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3) of the immunoglobulin, but does not include the variable regions of the heavy and light chains of the immunoglobulin and the light chain constant region 1 (CL1)). A native Fc region is a homodimer. In some embodiments, the term also encompasses variant Fc regions that have one or more changes compared to the native Fc region. The Fc region may be altered by amino acid substitution, addition, and / or deletion, linking of additional moieties, and / or alteration of native glycans. The term also encompasses Fc regions in which each of the constituent Fc domains is different. Examples of heterodimeric Fc regions include, but are not limited to, Fc regions generated using "knobs into holes" technology, e.g., as described in U.S. Pat. No. 8,216,805, or by SEED technology, as described in WO 2016 / 087650.

[0204] In some embodiments, the compound comprises an antibody Fc region competent in Fc receptor binding. An antibody Fc region is "capable of binding to an Fc receptor" if the antibody Fc region is capable of binding to at least one Fc receptor (including the FcγRI, FcγRII, and FcγRIII subclasses, allelic variants and alternatively spliced ​​forms of these receptors).

[0205] In some embodiments, the compound comprises an antibody Fc region that is incapable of binding to an Fc receptor. In some embodiments, the compound does not comprise an effector-capable antibody Fc region.

[0206] An "effector-capable" Fc region is an Fc region that has a functional binding capacity to proteins and / or cells of the immune system and mediates biological effects that are normally induced after an antibody binds to its corresponding antigen. Such biological effects include, by way of example, the ability to bind complement proteins (e.g., C1q), resulting in activation of the classical complement system leading to opsonization, and lysis of cellular pathogens (complement-dependent cytotoxicity, CDCC). Other biological effects are endocytosis of immune complexes, phagocytosis 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, modulation of immune system cell activation, or control of immunoglobulin production.

[0207] In some embodiments, the compound comprises an antibody Fc region with effector capabilities. In some embodiments, the compound does not comprise an antibody Fc region capable of inducing ADCC (antibody-dependent cellular cytotoxicity). In some embodiments, the compound comprises an antibody Fc region capable of inducing ADCC. In some embodiments, the compound comprises an antibody Fc region that is not capable of inducing ADCC.

[0208] In some embodiments, the compound is capable of inducing both FcγRIIIa signaling and positive (ie, activating NK cells) NKp30 signaling. In some embodiments, the compound is capable of specifically binding to NKp30 on NK cells. In some embodiments, the compound is capable of activating NK cells by binding to NKp30 on the NK cells.

[0209] Whether a compound or domain is capable of activating NK cells upon binding to NKp30 on NK cells can be determined as described in Example 1, section "NK cell activation assay." Whether this activation occurs upon binding to NKp30 on NK cells can be determined, for example, by performing control experiments on NK cells in which NKp30 is blocked by a competitor molecule that binds to NKp30 such that the protein domain is inaccessible to NKp30 on the NK cells.

[0210] Preferably, binding to NKp30 and activation of NK cells is assessed with said VHH antibody domain in the context of the complete compound. In some embodiments, the binding of the compound to NKp30 on an NK cell activates the NK cell. In some embodiments, the compound is an agonist of NKp30.

[0211] In some embodiments, the compound is capable of activating NK cells upon binding of said VHH antibody domain or fragment thereof to NKp30 on NK cells. In some embodiments, the binding of the compound to NKp30 on an NK cell activates the NK cell. In some embodiments, NK cell activation is determined by measuring the expression of the activation marker CD69 by flow cytometry.

[0212] In some embodiments, the compound is 51 It exhibits cytotoxic activity in a Cr release assay. In some embodiments, the 51 Cr release assay was performed for 4 h. 51 A Cr release assay was performed in which human PBMCs were used as effector cells at an effector-to-target cell (E:T) ratio of 80:1, and in which a higher percent lysis indicates improved cytotoxic activity. In some embodiments, the 51Cr release assays are performed as described in Repp et al., 2011.

[0213] As used herein, the reference "Repp et al., 2011" refers to the publication R. Repp et al., "Combined Fc-protein- and Fc-glyco-engineering of scFv-Fc fusion proteins synergistically enhances CD16a binding but does not further enhance NK-cell mediated ADCC", Journal of Immunological Methods (2011), vol. 373, p. 67-78. In some embodiments, binding of the compound to NKp30 on NK cells results in the release of interferon-γ (IFN-γ).

[0214] In some embodiments, the release of IFN-γ is measured as follows: isolated human NK cells are incubated overnight in medium containing 100 U / ml recombinant human interleukin-2, A431 cells are seeded in different wells and incubated for 3 h, compounds are added to a final concentration of 85 nM, followed by addition of NK cells at an E:T ratio of 5:1, and human IFN-γ in the culture supernatants after 24 h is analyzed by ELISA.

[0215] In some embodiments, binding of the compound to NKp30 on NK cells results in the release of tumor necrosis factor-α (TNF-α).

[0216] In some embodiments, the release of TNF-α is measured as follows: isolated human NK cells are incubated overnight in medium containing 100 U / ml recombinant human interleukin-2, A431 cells are seeded in different wells and incubated for 3 h, compounds are added to a final concentration of 85 nM, followed by addition of NK cells at an E:T ratio of 5:1, and human TNF-α in the culture supernatants after 24 h is analyzed by ELISA.

[0217] According to another aspect, the present disclosure relates to a pharmaceutical composition comprising a compound according to any one of the above described aspects or embodiments. Methods for preparing pharmaceutical compositions are known to those skilled in the art (Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), Pharmaceutical Press).

[0218] In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier, diluent, and / or excipient.

[0219] The term "pharmaceutical acceptable" specifies that the carrier, diluent, or excipient is a non-toxic, inert material that is compatible with other components of the pharmaceutical composition and is not harmful to the patient to whom the pharmaceutical composition is administered, so that it can be used in a pharmaceutical product. Substances suitable as carriers, diluents, or excipients in pharmaceutical compositions are known to those skilled in the art (Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), Pharmaceutical Press). The pharmaceutical composition may further include, for example, additional adjuvants, antioxidants, buffers, bulking agents, colorants, emulsifiers, fillers, flavoring agents, preservatives, stabilizers, suspending agents, and / or other conventional pharmaceutical auxiliary agents.

[0220] In some embodiments, the pharmaceutical composition further comprises at least one additional adjuvant, antioxidant, buffer, bulking agent, colorant, emulsifier, filler, flavoring agent, preservative, stabilizer, suspending agent, and / or other conventional pharmaceutical auxiliary. According to another aspect, the present disclosure relates to a compound according to any of the above described aspects or embodiments, or a pharmaceutical composition according to any of the above described aspects or embodiments, for use as a medicament.

[0221] According to another aspect, the present disclosure relates to a compound according to any of the above described aspects or embodiments, or a pharmaceutical composition according to any of the above described aspects or embodiments, for use in the treatment of cancer. According to another aspect, the present disclosure relates to a compound according to any of the above described aspects or embodiments, or a pharmaceutical composition according to any of the above described aspects or embodiments, for use in the treatment of malignant tumors.

[0222] In some embodiments, the compound / pharmaceutical composition is for use in treating humans. The production of medicaments containing the compounds of the present disclosure or pharmaceutical compositions according to the present disclosure can be carried out according to well-known pharmaceutical methods. Further details on the techniques for formulation and administration can be found, for example, in Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), Pharmaceutical Press.

[0223] As used herein, "treatment" of a disease and "treating" a disease refer to the process of providing a subject with medical treatment, e.g., the administration of a drug, such that the disease is alleviated, reduced, minimized, stopped, or even cured, and / or the chance of recurrence of the disease is reduced or prevented from recurring.

[0224] The use of compounds in the treatment of diseases is known to those skilled in the art (see, for example, Coats et al., Clinical Cancer Research (2019), vol. 25 (18), p. 5441-5448; Rudra, Bioconjugate Chemistry (2020), vol. 31 (3), p. 462-473). Thus, those skilled in the art know that the components of the compound, especially the targeting moiety, must be appropriately selected to enable successful treatment. For example, for the treatment of a particular cancer, the targeting moiety of the compound must be selected such that the binding of the targeting moiety to its target site directs the compound to the cancer (for example, by using an antibody component against a tumor-associated antigen that is specifically found on the surface of cancer cells). The cytotoxic effect 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 so that a desired additional treatment effect is achieved. For example, cytotoxic drugs for the treatment of cancer may additionally be included.

[0225] In another aspect, the disclosure relates to a method for treating a disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound according to any of the above described aspects or embodiments, or a pharmaceutical composition according to any of the above described aspects or embodiments.

[0226] "Therapeutically effective amount" refers to the amount of agent required to ameliorate the symptoms of a disease. The effective amount of active agent(s) (e.g., compounds according to the present disclosure) used for therapeutic treatment of a disease according to the present disclosure will vary depending on the mode of administration, age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as a "therapeutically effective" amount.

[0227] 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, the disease is cancer. In some embodiments, the disease is a malignant tumor.

[0228] As used herein, the term "cancer" refers to malignant neoplasms. Cancer may include hematological cancer or solid tumors. For example, cancer may be leukemia (e.g., acute myeloid leukemia (AML), acute monocytic leukemia, promyelocytic leukemia, eosinophilic leukemia, acute lymphoblastic leukemia (ALL), such as acute B-lymphoblastic leukemia (B-ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL)), or lymphoma (e.g., non-Hodgkin's 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 this disclosure, the term "cancer" refers to solid malignant tumors.

[0229] In some embodiments, the patient is a human. In another aspect, the present disclosure relates to the use of a compound according to any of the above described aspects or embodiments, or of a pharmaceutical composition according to any of the above described aspects or embodiments, for the manufacture of a medicament.

[0230] In another aspect, the present disclosure relates to the use of a compound according to any of the above described aspects or embodiments, or a pharmaceutical composition according to any of the above described aspects or embodiments, for the manufacture of a medicament for the treatment of cancer. In another aspect, the present disclosure relates to the use of a compound according to any of the above described aspects or embodiments, or a pharmaceutical composition according to any of the above described aspects or embodiments, for the manufacture of a medicament for the treatment of a malignant tumor.

[0231] In some embodiments, the medicament is prepared for administration to a human. The following aspects relate to either a compound or pharmaceutical composition for use in medical treatment, a method for treating a disease in a patient in need thereof, a use for the manufacture of a medicament, or any of those aspects described above.

[0232] In some embodiments, the cancer or malignancy is a human disease. The following aspects relate to any of the compounds, pharmaceutical compositions, 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 those aspects described above.

[0233] In some embodiments, the compound comprises a sequence represented by SEQ ID NO:1 (or a fragment thereof) and has one or more amino acid substitutions compared to the sequence of SEQ ID NO:1, where if such amino acid substitutions are located outside the CDRs, the amino acid substitution(s) are selected from the following: D1E, Q5V, L11V, T24A, E44G, H45L, G47W, F70I, V79L, K87R, P88A, Q116L.

[0234] In some embodiments, the compound comprises a sequence represented by SEQ ID NO:1 (or a fragment thereof) with at least the following amino acid substitutions: D1E, Q5V, L11V, V79L, K87R, P88A, Q116L; and optionally, one or more additional amino acid substitutions compared to the sequence of SEQ ID NO:1, where if such additional amino acid substitutions are located outside the CDRs, such additional amino acid substitution(s) are selected from the following: T24A, E44G, H45L, F70I.

[0235] In some embodiments, the compound comprises a sequence represented by SEQ ID NO:1 (or a fragment thereof) with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] .

[0236] In some embodiments, the compound comprises a sequence represented by SEQ ID NO:1 (or a fragment thereof) with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] ; and with one or more additional amino acid substitutions compared to the sequence of SEQ ID NO:1, wherein the amino acid substitution(s) are selected from the following: D1E, Q5V, L11V, T24A, E44G, H45L, G47W, F70I, V79L, K87R, P88A, Q116L.

[0237] In some embodiments, the compound comprises a sequence represented by SEQ ID NO:1 (or a fragment thereof) with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] ; and with amino acid substitutions D1E, Q5V, L11V, V79L, K87R, P88A, Q116L; and optionally with one or more additional amino acid substitutions compared to the sequence of SEQ ID NO:1, wherein the additional amino acid substitution(s) are selected from the following: T24A, E44G, H45L, F70I.

[0238] In some embodiments, the compound comprises a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or a fragment thereof. In some embodiments, the compound has a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or a fragment thereof. In some embodiments, the compound has a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, or a fragment thereof. In some embodiments, the compound has a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:77, or a fragment thereof. In some embodiments, the compound comprises a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, or a fragment thereof. In some embodiments, the compound comprises a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:78, or a fragment thereof. In some embodiments, the compound comprises a sequence defined by SEQ ID NO: 77, or a fragment thereof. In some embodiments, the compound comprises a sequence defined by SEQ ID NO: 78, or a fragment thereof.

[0239] In some embodiments, the compound comprises a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or a fragment thereof; with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] .

[0240] In some embodiments, the compound comprises a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or a fragment thereof; with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] .

[0241] In some embodiments, the compound comprises a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, or a fragment thereof; with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] .

[0242] In some embodiments, the compound comprises a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:77, or a fragment thereof; with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] .

[0243] In some embodiments, the compound comprises a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, or a fragment thereof; with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] .

[0244] In some embodiments, the compound comprises a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:78, or a fragment thereof; with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] .

[0245] In some embodiments, the compound comprises a sequence defined by SEQ ID NO: 77 or a fragment thereof, with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] .

[0246] In some embodiments, the compound comprises a sequence defined by SEQ ID NO: 78 or a fragment thereof, with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] .

[0247] In some embodiments, the compound comprises a sequence represented by SEQ ID NO:2 (or a fragment thereof) and has one or more amino acid substitutions compared to the sequence of SEQ ID NO:2, where if such amino acid substitutions are located outside the CDRs, the amino acid substitution(s) are selected from the following: D1E, Q5V, L11V, A14P, V24A, F37V, A44G, R45L, S47W, V79L, K87R, P88A, Q118L.

[0248] In some embodiments, the compound comprises a sequence represented by SEQ ID NO:2 (or a fragment thereof) with amino acid substitutions D1E, Q5V, L11V, K87R, P88A, Q118L; and optionally, one or more additional amino acid substitutions compared to the sequence of SEQ ID NO:2, where if such amino acid substitutions are located outside the CDRs, such additional amino acid substitution(s) are selected from the following: A14P, V24A, A44G, R45L, S47W, V79L.

[0249] In some embodiments, the compound comprises a sequence represented by SEQ ID NO:2 (or a fragment thereof) with amino acid substitutions D1E, Q5V, L11V, A14P, V24A, K87R, P88A, Q118L; and optionally, one or more additional amino acid substitutions compared to the sequence of SEQ ID NO:2, where if such amino acid substitutions are located outside the CDRs, such additional amino acid substitution(s) are selected from the following: A44G, R45L, S47W, V79L.

[0250] In some embodiments, the compound comprises a sequence represented by SEQ ID NO:2 (or a fragment thereof) with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] .

[0251] In some embodiments, the compound comprises a sequence represented by SEQ ID NO:2 (or a fragment thereof) with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] ; and with one or more additional amino acid substitutions compared to the sequence of SEQ ID NO:2, wherein the amino acid substitution(s) are selected from the following: D1E, Q5V, L11V, A14P, V24A, F37V, A44G, R45L, S47W, V79L, K87R, P88A, Q118L.

[0252] In some embodiments, the compound comprises a sequence represented by SEQ ID NO:2 (or a fragment thereof) with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] ; and with amino acid substitutions D1E, Q5V, L11V, K87R, P88A, Q118L; and optionally with one or more additional amino acid substitutions compared to the sequence of SEQ ID NO:2, wherein the additional amino acid substitution(s) are selected from the following: A14P, V24A, A44G, R45L, S47W, V79L.

[0253] In some embodiments, the compound comprises a sequence represented by SEQ ID NO:2 (or a fragment thereof) with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] ; and with amino acid substitutions D1E, Q5V, L11V, A14P, V24A, K87R, P88A, Q118L; and optionally with one or more additional amino acid substitutions compared to the sequence of SEQ ID NO:2, wherein the additional amino acid substitution(s) are selected from the following: A44G, R45L, S47W, V79L.

[0254] In some embodiments, the compound comprises a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:94, or a fragment thereof.

[0255] In some embodiments, the compound comprises a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:94, or a fragment thereof.

[0256] In some embodiments, the compound comprises a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:94, or a fragment thereof; with an additional modification of (b) according to any one of items [1]-

[11] , or with an additional modification of (c) according to any one of items [1] or

[12] -

[22] .

[0257] In some embodiments, the compound comprises a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:94, or a fragment thereof; with an additional modification (b) according to any one of items [1]-

[11] , or with an additional modification (c) according to any one of items [1] or

[12] -

[22] .

[0258] In some embodiments, the VHH antibody domain comprises the VHH sequence VHH1 as shown in the Table of VHH Sequences or a fragment thereof, with an additional modification of (B) or (C), wherein said modification of (B) or (C) consists of one or more amino acid substitutions compared to the sequence of SEQ ID NO:1, wherein said amino acid substitution(s) is / are selected from the following: D1E, Q5V, L11V, T24A, E44G, H45L, G47W, F70I, V79L, K87R, P88A, Q116L.

[0259] In some embodiments, the VHH antibody domain comprises the VHH sequence VHH1 as shown in the Table of VHH sequences or a fragment thereof, with an additional modification of (B) or (C), wherein the modification of (B) or (C) consists of the amino acid substitutions D1E, Q5V, L11V, V79L, K87R, P88A, Q116L compared to the sequence of SEQ ID NO:1; and optionally one or more further amino acid substitutions compared to the sequence of SEQ ID NO:1, wherein the further amino acid substitution(s) are selected from the following: T24A, E44G, H45L, F70I.

[0260] In some embodiments, the VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or a fragment thereof.

[0261] In some embodiments, the VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or a fragment thereof. In some embodiments, the VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, or a fragment thereof. In some embodiments, the VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:77, or a fragment thereof. In some embodiments, the VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, or a fragment thereof. In some embodiments, wherein said VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:78, or a fragment thereof.

[0262] In some embodiments, the VHH antibody domain comprises the VHH sequence VHH1 as shown in the Table of VHH Sequences or a fragment thereof, with an additional modification of (B) or (C), wherein said modification of (B) or (C) consists of one or more amino acid substitutions compared to the sequence of SEQ ID NO:2, wherein said amino acid substitution(s) is / are selected from the following: D1E, Q5V, L11V, A14P, V24A, F37V, A44G, R45L, S47W, V79L, K87R, P88A, Q118L.

[0263] In some embodiments, the VHH antibody domain comprises the VHH sequence VHH1 as shown in the Table of VHH sequences or a fragment thereof, with an additional modification of (B) or (C), wherein the modification of (B) or (C) consists of the amino acid substitutions D1E, Q5V, L11V, K87R, P88A, Q118L compared to the sequence of SEQ ID NO:2; and optionally one or more further amino acid substitutions compared to the sequence of SEQ ID NO:2, wherein the further amino acid substitution(s) is / are selected from the following: A14P, V24A, A44G, R45L, S47W, V79L.

[0264] In some embodiments, the VHH antibody domain comprises the VHH sequence VHH1 as shown in the Table of VHH sequences or a fragment thereof, with an additional modification of (B) or (C), wherein the modification of (B) or (C) consists of the amino acid substitutions D1E, Q5V, L11V, A14P, V24A, K87R, P88A, Q118L compared to the sequence of SEQ ID NO:2; and optionally one or more further amino acid substitutions compared to the sequence of SEQ ID NO:2, wherein the further amino acid substitution(s) are selected from the following: A44G, R45L, S47W, V79L.

[0265] In some embodiments, the VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:94, or a fragment thereof.

[0266] In some embodiments, the VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:94, or a fragment thereof.

[0267] In some embodiments, the compound binds to the recombinant extracellular domain of NKp30 (NKp30-ECD, SEQ ID NO: 66) in KB buffer (PBS + 0.1% Tween-20 + 1% BSA) by BLI (BioLayer Interferometry) at least about 1 x 10 6 M -1 It binds to NKp30 with an affinity of In some embodiments, the compound binds to the recombinant extracellular domain of NKp30 (NKp30-ECD, SEQ ID NO: 66) in KB buffer (PBS + 0.1% Tween-20 + 1% BSA) by BLI (BioLayer Interferometry) at least about 5×10 6 M -1 It binds to NKp30 with an affinity of In some embodiments, the compound binds to the recombinant extracellular domain of NKp30 (NKp30-ECD, SEQ ID NO: 66) in KB buffer (PBS + 0.1% Tween-20 + 1% BSA) by BLI (BioLayer Interferometry) at least about 1 x 10 7 M -1 It binds to NKp30 with an affinity of In some embodiments, the compound binds to the recombinant extracellular domain of NKp30 (NKp30-ECD, SEQ ID NO: 66) in KB buffer (PBS + 0.1% Tween-20 + 1% BSA) by BLI (BioLayer Interferometry) at least about 5×10 87 M -1 It binds to NKp30 with an affinity of In some embodiments, the compound binds to the recombinant extracellular domain of NKp30 (NKp30-ECD, SEQ ID NO: 66) in KB buffer (PBS + 0.1% Tween-20 + 1% BSA) by BLI (BioLayer Interferometry) at least about 1 x 10 8 M -1 It binds to NKp30 with an affinity of

[0268] Also disclosed in relation to the above subject matter: [1] A compound comprising a VHH antibody domain or a fragment thereof, (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2, and CDR3 of one of VHH1 to VHH16 as shown in the CDR table below; (b) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with a modification, wherein the modification is that at least one sequence 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 a modification, wherein the modification is - substitutions, additions or deletions of up to 3 amino acids in CDR1, - replacement, addition or deletion of up to 3 amino acids in CDR2, and / or - substitutions, additions, or deletions of up to 3 amino acids in the CDR3; CDR Table: [Table F-1]

[0269] [Table F-2]

[0270] [2] The compound according to item [1], wherein the modification in (b) is that the CDR1 and / or CDR2 sequences, but not the CDR3 sequence, are humanized.

[0271] [3] The compound of any one of items [1] or [2], wherein the modification in (b) is that the CDR1 sequence, but not the CDR2 and CDR3 sequences, is humanized.

[0272] [4] The compound of any one of items [1] or [2], wherein the modification in (b) is that the CDR2 sequence, but not the CDR1 and CDR3 sequences, is humanized.

[0273] [5] The compound of any one of items [1] to [4], wherein the modification in (b) is that one, but not two or more, of CDR1, CDR2, and CDR3 sequences is humanized.

[0274] [6] The compound of any one of items [1] to [5], wherein the humanization of the CDR(s) is by replacing at least one amino acid in the sequence of the CDR with a corresponding amino acid of a human VH domain.

[0275] [7] The compound of any one of items [1] to [6], wherein the humanization of the CDR(s) is by replacing up to 3 amino acids in the sequence of the CDR with corresponding amino acids of a human VH domain.

[0276] [8] The compound of any one of items [1] to [6], wherein the humanization of the CDR(s) is by replacing up to three amino acids in the CDR1 and / or CDR2 sequence and up to one amino acid in the CDR3 sequence with corresponding amino acids from a human VH domain.

[0277] [9] The compound of any one of items [1] to [6], wherein the humanization of the CDR(s) is by replacing up to two amino acids in the sequence of the CDR with corresponding amino acids of a human VH domain.

[0278]

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

[0279]

[11] The compound according to any one of items [1] to

[10] , wherein the humanization of the CDR(s) is by replacing one amino acid in the sequence of the CDR with a corresponding amino acid from a human VH domain.

[0280]

[12] The modification in (c) is - substitutions, additions or deletions of up to 3 amino acids in CDR1, - replacement, addition or deletion of up to 3 amino acids in CDR2, and / or - A compound according to any one of items [1] to

[11] , which is a substitution, addition, or deletion of up to one amino acid in CDR3.

[0281]

[13] The modification in (c) is - substitution, addition or deletion of up to 2 amino acids in CDR1, - replacement, addition or deletion of up to 2 amino acids in CDR2, and / or - any one of the compounds of items [1] to

[11] , which is a substitution, addition, or deletion of up to two amino acids in CDR3;

[0282]

[14] The modification in (c) is - replacement, addition or deletion of up to 2 amino acids in CDR1; - a substitution, addition or deletion of up to 2 amino acids in CDR2; and / or - A compound according to any one of items [1] to

[11] , which is a substitution, addition, or deletion of up to one amino acid in CDR3.

[0283]

[15] The modification in (c) is - replacement, addition or deletion of up to 2 amino acids in CDR1, and / or - a maximum of two amino acid replacements, additions, or deletions in CDR2; wherein the CDR3 sequence is unmodified, a compound according to any one of items [1] to

[11] .

[0284]

[16] The modification in (c) is - a maximum of two amino acid substitutions, additions or deletions in CDR1, wherein the sequences of CDR2 and CDR3 are unmodified, a compound according to any one of items [1] to

[11] .

[0285]

[17] The modification in (c) is - a maximum of two amino acid substitutions, additions or deletions in CDR2, wherein the sequences of CDR1 and CDR3 are unmodified, a compound according to any one of items [1] to

[11] .

[0286]

[18] The modification in (c) is - replacement, addition or deletion of up to one amino acid in CDR1; - a substitution, addition or deletion of up to one amino acid in CDR2; and / or - A compound according to any one of items [1] to

[11] , which is a substitution, addition, or deletion of up to one amino acid in CDR3.

[0287]

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

[11] .

[0288]

[20] The modification in (c) is - a substitution, addition or deletion of one amino acid in CDR1, wherein the sequences of CDR2 and CDR3 are unmodified, a compound according to any one of items [1] to

[11] .

[0289]

[21] The modification in (c) is - a substitution, addition or deletion of one amino acid in CDR2, wherein the sequences of CDR1 and CDR3 are unmodified, a compound according to any one of items [1] to

[11] .

[0290]

[22] A compound according to any one of items [1] to

[21] , wherein the modification in (c) includes only amino acid replacement and does not include amino acid addition or deletion.

[0291]

[23] A compound comprising a VHH antibody domain or a fragment thereof, (A) the VHH antibody domain comprises any one of VHH1 to VHH16 shown in the VHH sequence table below; (B) the VHH antibody domain comprises a VHH sequence as defined in (A) with a modification, wherein the modification is that the sequence is humanized; (C) the VHH antibody domain comprises a VHH sequence as defined in (A) with a modification, wherein the modification is a replacement, addition or deletion of up to 25 amino acids; or (D) the VHH antibody domain comprises a VHH sequence that is at least 75% identical to the VHH sequence set forth in (A); Table of VHH sequences: [Table G-1]

[0292] [Table G-2]

[0293] [Table G-3]

[0294]

[24] A compound comprising a VHH antibody domain or a fragment thereof, (A) the VHH antibody domain consists of any one of VHH1 to VHH16 shown in the VHH sequence table; (B) the VHH antibody domain consists of a VHH sequence as defined in (A) with a modification, wherein the modification is that the sequence is humanized; (C) the VHH antibody domain consists of a VHH sequence as defined in (A) with a modification, wherein the modification is a replacement, addition or deletion of up to 25 amino acids; or (D) The VHH antibody domain consists of a VHH sequence that is at least 75% identical to the VHH sequence mentioned in (A).

[0295]

[25] The compound of any one of items

[23] or

[24] , wherein the fragment of the VHH antibody domain comprises at least 75% of the amino acids of the sequence of the VHH antibody domain.

[0296]

[26] The compound of any one of items

[23] or

[24] , wherein the fragment of the VHH antibody domain comprises at least 80% of the amino acids of the sequence of the VHH antibody domain.

[0297]

[27] The compound of any one of items

[23] or

[24] , wherein the fragment of the VHH antibody domain comprises at least 85% of the amino acids of the sequence of the VHH antibody domain.

[0298]

[28] The compound of any one of items

[23] or

[24] , wherein the fragment of the VHH antibody domain comprises at least 90% of the amino acids of the sequence of the VHH antibody domain.

[0299]

[29] The compound of any one of items

[23] or

[24] , wherein the fragment of the VHH antibody domain comprises at least 95% of the amino acids of the sequence of the VHH antibody domain.

[0300]

[30] The compound of any one of items

[23] or

[24] , wherein the fragment of the VHH antibody domain comprises at least 98% of the amino acids of the sequence of the VHH antibody domain.

[0301]

[31] The compound of any one of items

[23] or

[24] , wherein the fragment of the VHH antibody domain comprises at least 99% of the amino acids of the sequence of the VHH antibody domain.

[0302]

[32] The compound of any one of items

[23] to

[31] , wherein the fragment of the VHH antibody domain comprises the complementarity determining regions CDR1, CDR2, and CDR3.

[0303]

[33] The compound of any one of items

[23] to

[32] , wherein the fragment of the VHH antibody domain comprises at least a sequence from the N-terminus of CDR1 to the C-terminus of CDR3 of the VHH antibody domain.

[0304]

[34] A compound according to any one of items

[23] to

[33] , wherein the fragment of the VHH antibody domain in (A) comprises all of the complementarity determining regions (CDRs) of the VHH antibody domain.

[0305]

[35] The compound of any one of items

[23] to

[34] , wherein the humanization of the sequence in (B) is by replacing at least one amino acid of the sequence with a corresponding amino acid of a human VH (variable heavy) domain.

[0306]

[36] The compound of any one of items

[23] to

[35] , wherein the humanization of the sequence in (B) is by replacing up to 25 amino acids of the sequence by corresponding amino acids of a human VH domain.

[0307]

[37] The compound of any one of items

[23] to

[35] , wherein the humanization of the sequence in (B) is by replacing up to 20 amino acids of the sequence by corresponding amino acids of a human VH domain.

[0308]

[38] The compound of any one of items

[23] to

[35] , wherein the humanization of the sequence in (B) is by replacing up to 15 amino acids of the sequence with corresponding amino acids of a human VH domain.

[0309]

[39] The compound of any one of items

[23] to

[35] , wherein the humanization of the sequence in (B) is by replacing up to 10 amino acids of the sequence by corresponding amino acids of a human VH domain.

[0310]

[40] The compound of any one of items

[23] to

[35] , wherein the humanization of the sequence in (B) is by replacing up to 5 amino acids of the sequence by corresponding amino acids of a human VH domain.

[0311]

[41] The compound of any one of items

[23] to

[35] , wherein the humanization of the sequence in (B) is by replacing up to three amino acids of the sequence by corresponding amino acids of a human VH domain.

[0312]

[42] The compound of any one of items

[23] to

[35] , wherein the humanization of the sequence in (B) is by replacing a maximum of two amino acids of the sequence by corresponding amino acids of a human VH domain.

[0313]

[43] The compound of any one of items

[23] to

[35] , wherein the humanization of the sequence in (B) is by replacing one amino acid of the sequence with a corresponding amino acid of a human VH domain.

[0314]

[44] The compound of any one of items

[23] to

[43] , wherein the humanization in (B) is within the framework regions of the VHH antibody domain and / or within the CDRs of the VHH antibody domain.

[0315]

[45] The compound of any one of items

[23] to

[43] , wherein the humanization in (B) is within the framework region of the VHH antibody domain but not within the CDR of the VHH antibody domain.

[0316]

[46] The compound of any one of items

[23] to

[43] , wherein the humanization in (B) is within the CDRs of the VHH antibody domain but not within the framework regions of the VHH antibody domain.

[0317]

[47] The compound of any one of items

[23] to

[44] or

[46] , wherein the humanization within the CDRs of the VHH antibody domain in (B) is within CDR1, CDR2, and / or CDR3.

[0318]

[48] ​​A compound according to any one of items

[23] to

[44] or

[46] to

[47] , wherein the humanization within the CDRs of the VHH antibody domain in (B) is within CDR1 and / or CDR2.

[0319]

[49] A compound according to any one of items

[23] to

[44] or

[46] to

[48] , wherein the humanization within the CDR of the VHH antibody domain in (B) is within CDR1.

[0320]

[50] A compound according to any one of items

[23] to

[44] or

[46] to

[49] , wherein the humanization within the CDR of the VHH antibody domain in (B) is within CDR2.

[0321]

[51] The compound of any one of items

[23] to

[50] , wherein the humanization within the CDR of the VHH antibody domain in (B) is not within CDR3.

[0322]

[52] A compound according to any one of items

[23] to

[51] , wherein the modification in (C) is a substitution, addition, or deletion of up to 20 amino acids.

[0323]

[53] A compound according to any one of items

[23] to

[51] , wherein the modification in (C) is a substitution, addition, or deletion of up to 15 amino acids.

[0324]

[54] A compound according to any one of items

[23] to

[51] , wherein the modification in (C) is a substitution, addition, or deletion of up to 10 amino acids.

[0325]

[55] A compound according to any one of items

[23] to

[51] , wherein the modification in (C) is a substitution, addition, or deletion of up to five amino acids.

[0326]

[56] A compound according to any one of items

[23] to

[51] , wherein the modification in (C) is a substitution, addition, or deletion of up to three amino acids.

[0327]

[57] A compound according to any one of items

[23] to

[51] , wherein the modification in (C) is a substitution, addition, or deletion of up to two amino acids.

[0328]

[58] A compound according to any one of items

[23] to

[51] , wherein the modification in (C) is a replacement, addition, or deletion of one amino acid.

[0329]

[59] A compound according to any one of items

[23] to

[58] , wherein the modification in (c) involves only amino acid replacement and does not involve addition or deletion of amino acids.

[0330]

[60] A compound according to any one of items

[23] to

[59] , wherein the VHH antibody domain or fragment thereof in (A) comprises the complementarity determining regions CDR1, CDR2, and CDR3 of one of VHH1 to VHH16 as shown in the CDR table.

[0331]

[61] In (B) to (D), (a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2, and CDR3 of one of VHH1 to VHH16 as shown in the CDR Table; (b) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with a modification, wherein the modification is that at least one sequence of CDR1, CDR2 and CDR3 is humanized; (c) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with a modification, wherein the modification is - substitutions, additions or deletions of up to 3 amino acids in CDR1, - replacement, addition or deletion of up to 3 amino acids in CDR2, and / or - A compound according to any one of items

[23] to

[60] , which is a substitution, addition, or deletion of up to three amino acids in CDR3.

[0332]

[62] The compound of item

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

[0333]

[63] The compound of any one of items

[61] or

[62] , wherein the modification in (b) is that the CDR1 sequence but not the CDR2 and CDR3 sequences are humanized.

[0334]

[64] The compound of any one of items

[61] or

[62] , wherein the modification in (b) is that the CDR2 sequence but not the CDR1 and CDR3 sequences are humanized.

[0335]

[65] The compound of any one of items

[61] to

[64] , wherein the modification in (b) is that the sequence of one, but not two or more, of CDR1, CDR2, and CDR3 is humanized.

[0336]

[66] The compound of any one of items

[61] to

[65] , wherein the humanization of the CDR(s) is by replacing at least one amino acid in the sequence of the CDR with a corresponding amino acid from a human VH domain.

[0337]

[67] The compound of any one of items

[61] to

[66] , wherein the humanization of the CDR(s) is by replacing up to three amino acids in the sequence of the CDR with corresponding amino acids of a human VH domain.

[0338]

[68] The compound of any one of items

[61] to

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

[0339]

[69] The compound of any one of items

[61] to

[66] , wherein the humanization of the CDR(s) is by replacing a maximum of two amino acids in the sequence of the CDR by corresponding amino acids of a human VH domain.

[0340]

[70] The compound of any one of items

[61] to

[66] , wherein the humanization of the CDR(s) is by replacing a maximum of two amino acids in the sequence of CDR1 and / or CDR2 and a maximum of one amino acid in the sequence of CDR3 by the corresponding amino acid of a human VH domain.

[0341]

[71] The compound of any one of items

[61] to

[70] , wherein the humanization of the CDR(s) is by replacing one amino acid in the sequence of the CDR with a corresponding amino acid from a human VH domain.

[0342]

[72] The modification in (c) is - substitutions, additions or deletions of up to 3 amino acids in CDR1, - replacement, addition or deletion of up to 3 amino acids in CDR2, and / or - Any one of the compounds of items

[61] to

[71] , which is a substitution, addition, or deletion of up to two amino acids in CDR3.

[0343]

[73] The modification in (c) is - substitutions, additions or deletions of up to 3 amino acids in CDR1, - replacement, addition or deletion of up to 3 amino acids in CDR2, and / or - Any one of the compounds of items

[61] to

[71] , which is a substitution, addition, or deletion of up to one amino acid in CDR3.

[0344]

[74] The modification in (c) is - substitutions, additions or deletions of up to 3 amino acids in CDR1, and / or - a substitution, addition or deletion of up to 3 amino acids in CDR2, wherein the CDR3 sequence is unmodified, a compound according to any one of items

[61] to

[71] .

[0345]

[75] The modification in (c) is - substitution, addition or deletion of up to 2 amino acids in CDR1, - replacement, addition or deletion of up to 2 amino acids in CDR2, and / or - any one of the compounds under items

[61] to

[71] , which is a substitution, addition, or deletion of up to two amino acids in CDR3;

[0346]

[76] The modification in (c) is - replacement, addition or deletion of up to 2 amino acids in CDR1; - a substitution, addition or deletion of up to 2 amino acids in CDR2; and / or - Any one of the compounds of items

[61] to

[71] , which is a substitution, addition, or deletion of up to one amino acid in CDR3.

[0347]

[77] The modification in (c) is - replacement, addition or deletion of up to 2 amino acids in CDR1, and / or - a maximum of two amino acid replacements, additions, or deletions in CDR2; wherein the CDR3 sequence is unmodified, a compound according to any one of items

[61] to

[71] .

[0348]

[78] The modification in (c) is - a maximum of two amino acid substitutions, additions or deletions in CDR1, wherein the sequences of CDR2 and CDR3 are unmodified, a compound according to any one of items

[61] to

[71] .

[0349]

[79] The modification in (c) is - a maximum of two amino acid substitutions, additions or deletions in CDR2, wherein the sequences of CDR1 and CDR3 are unmodified, a compound according to any one of items

[61] to

[71] .

[0350]

[80] The modification in (c) is - replacement, addition or deletion of up to one amino acid in CDR1; - a substitution, addition or deletion of up to one amino acid in CDR2; and / or - Any one of the compounds of items

[61] to

[71] , which is a substitution, addition, or deletion of up to one amino acid in CDR3.

[0351]

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

[61] to

[71] .

[0352]

[82] The modification in (c) is - a substitution, addition or deletion of one amino acid in CDR1, wherein the sequences of CDR2 and CDR3 are unmodified, a compound according to any one of items

[61] to

[71] .

[0353]

[83] The modification in (c) is - a substitution, addition or deletion of one amino acid in CDR2, wherein the sequences of CDR1 and CDR3 are unmodified, a compound according to any one of items

[61] to

[71] .

[0354]

[84] A compound according to any one of items

[61] to

[83] , wherein the modification in (c) involves only amino acid replacement and does not involve addition or deletion of amino acids.

[0355]

[85] The compound according to any one of items [1] to

[84] , wherein the compound comprises a VHH antibody domain (and not just a fragment of a VHH antibody domain).

[0356]

[86] The compound according to any one of items [1] to

[84] , wherein the compound consists of a fragment of a VHH antibody domain (not a full-length VHH antibody domain).

[0357]

[87] The compound according to any one of items [1] to

[84] or

[86] , wherein the fragment consists of at least 100 amino acids.

[0358]

[88] The compound according to any one of items [1] to

[84] or

[86] , wherein the fragment consists of at least 105 amino acids.

[0359]

[89] The compound according to any one of items [1] to

[84] or

[86] , wherein the fragment consists of at least 110 amino acids.

[0360]

[90] The compound according to any one of items [1] to

[84] or

[86] , wherein the fragment consists of at least 115 amino acids.

[0361]

[91] The compound according to any one of items [1] to

[90] , wherein the compound is capable of specifically binding to NKp30.

[0362]

[92] The compound according to any one of items [1] to

[91] , wherein the VHH antibody domain or a fragment thereof is capable of specifically binding to NKp30.

[0363]

[93] The VHH antibody domain or fragment thereof is injected into recombinant human NKp30 at 1x10 -6 A compound according to any one of items [1] to

[92] that binds at or stronger than the KD of M.

[0364]

[94] The VHH antibody domain or fragment thereof is injected into recombinant human NKp30 at 1x10 -7 A compound according to any one of items [1] to

[92] that binds at or stronger than the KD of M.

[0365]

[95] The VHH antibody domain or fragment thereof is injected into recombinant human NKp30 at 1x10 -8 A compound according to any one of items [1] to

[92] that binds at or stronger than the KD of M.

[0366]

[96] The VHH antibody domain or fragment thereof is injected into recombinant human NKp30 at 1x10 -9 A compound according to any one of items [1] to

[92] that binds at or stronger than the KD of M.

[0367]

[97] The VHH antibody domain or fragment thereof is injected into recombinant human NKp30 at 1x10 4 k greater than or equal to (1 / Ms) on A compound according to any one of items [1] to

[96] , which is bonded at

[0368]

[98] The VHH antibody domain or fragment thereof is injected into recombinant human NKp30 at 1x10 5 k greater than or equal to (1 / Ms) on A compound according to any one of items [1] to

[96] , which is bonded at

[0369]

[99] The VHH antibody domain or fragment thereof is injected into recombinant human NKp30 at 5x10 -3 k less than (1 / Ms) off A compound according to any one of items [1] to

[98] , which is bonded at

[0370] The VHH antibody domain or fragment thereof was injected into recombinant human NKp30 at 1x10 -3 k less than (1 / Ms) off A compound according to any one of items [1] to

[98] , which is bonded at

[0371] The VHH antibody domain or fragment thereof was injected into recombinant human NKp30 at 1x10 -4 k less than (1 / Ms) off A compound according to any one of items [1] to

[98] , which is bonded at

[0372] The VHH antibody domain or fragment thereof was injected into recombinant human NKp30 at 1x10 -5 k less than (1 / Ms) off A compound according to any one of items [1] to

[98] , which is bonded at

[0373] The VHH antibody domain or fragment thereof was injected into recombinant human NKp30 at 1x10 -6 k less than (1 / Ms) offA compound according to any one of items [1] to

[98] , which is bonded at

[0374] The VHH antibody domain or fragment thereof was injected into recombinant human NKp30 at 1x10 -7 k less than (1 / Ms) off A compound according to any one of items [1] to

[98] , which is bonded at

[0375]

[0105] A compound according to any one of items [1] to

[0104] , wherein the VHH antibody domain or fragment thereof in (b) and (c) binds to recombinant human NKp30 with an affinity (KD value) that is not at least 5-fold weaker than the binding of the corresponding unmodified VHH antibody domain.

[0376]

[0106] A compound according to any one of items [1] to

[0104] , wherein the VHH antibody domain or fragment thereof in (b) and (c) binds to recombinant human NKp30 with an affinity (KD value) that is not more than two-fold weaker than the binding of the corresponding unmodified VHH antibody domain.

[0377]

[0107] A compound according to any one of items [1] to

[0104] , wherein the VHH antibody domain or fragment thereof in (b) and (c) binds to recombinant human NKp30 with an affinity (KD value) that is not at least 1.5-fold weaker than the binding of the corresponding unmodified VHH antibody domain.

[0378]

[0108] A compound according to any one of items [1] to

[0107] , wherein the VHH antibody domain or fragment thereof in (b) and (c) binds to recombinant human NKp30 with an affinity (KD value) that is not more than 5-fold stronger than the binding of the corresponding unmodified VHH antibody domain.

[0379]

[0109] A compound according to any one of items [1] to

[0107] , wherein the VHH antibody domain or fragment thereof in (b) and (c) binds to recombinant human NKp30 with an affinity (KD value) that is not more than two-fold stronger than the binding of the corresponding unmodified VHH antibody domain.

[0380]

[0110] A compound according to any one of items [1] to

[0107] , wherein the VHH antibody domain or fragment thereof in (b) and (c) binds to recombinant human NKp30 with an affinity (KD value) that is not more than 1.5 times stronger than the binding of the corresponding unmodified VHH antibody domain.

[0381]

[0111] A compound according to any one of items

[23] to

[0110] , wherein the VHH antibody domains in (B) and (C) bind to recombinant human NKp30 with an affinity (KD value) that is not at least 5-fold weaker than the binding of the corresponding unmodified VHH antibody domain.

[0382]

[0112] A compound according to any one of items

[23] to

[0110] , wherein the VHH antibody domains in (B) and (C) bind to recombinant human NKp30 with an affinity (KD value) that is not more than two-fold weaker than the binding of the corresponding unmodified VHH antibody domain.

[0383]

[0113] A compound according to any one of items

[23] to

[0110] , wherein the VHH antibody domains in (B) and (C) bind to recombinant human NKp30 with an affinity (KD value) that is not at least 1.5-fold weaker than the binding of the corresponding unmodified VHH antibody domain.

[0384]

[0114] A compound according to any one of items

[23] to

[0113] , wherein the VHH antibody domains in (B) and (C) bind to recombinant human NKp30 with an affinity (KD value) that is not more than 5-fold stronger than the binding of the corresponding unmodified VHH antibody domain.

[0385]

[0115] A compound according to any one of items

[23] to

[0113] , wherein the VHH antibody domains in (B) and (C) bind to recombinant human NKp30 with an affinity (KD value) that is not more than two-fold stronger than the binding of the corresponding unmodified VHH antibody domain.

[0386]

[0116] A compound according to any one of items

[23] to

[0113] , wherein the VHH antibody domain in (B) and (C) binds to recombinant human NKp30 with an affinity (KD value) that is not more than 1.5-fold stronger than the binding of the corresponding unmodified VHH antibody domain.

[0387]

[0117] A compound according to any one of items

[23] to

[0116] , in which the binding affinity (KD value) of the VHH antibody domain in (D) to human NKp30 is not more than 5 times weaker than the binding affinity (KD value) of a VHH antibody domain consisting of a sequence from the table of VHH sequences having the highest sequence identity to the sequence of the VHH antibody domain in (D) to human NKp30.

[0388]

[0118] A compound according to any one of items

[23] to

[0116] , in which the binding affinity (KD value) of the VHH antibody domain in (D) to human NKp30 is not more than two times weaker than the binding affinity (KD value) of a VHH antibody domain consisting of a sequence from the table of VHH sequences having the highest sequence identity to the sequence of the VHH antibody domain in (D) to human NKp30.

[0389]

[0119] A compound according to any one of items

[23] to

[0116] , in which the binding affinity (KD value) of the VHH antibody domain in (D) to human NKp30 is not more than 1.5 times weaker than the binding affinity (KD value) of a VHH antibody domain consisting of a sequence from the table of VHH sequences having the highest sequence identity to the sequence of the VHH antibody domain in (D).

[0390]

[0120] A compound according to any one of items

[23] to

[0119] , in which the binding affinity (KD value) of the VHH antibody domain in (D) to human NKp30 is not more than 5 times stronger than the binding affinity (KD value) of a VHH antibody domain consisting of a sequence from the table of VHH sequences having the highest sequence identity to the sequence of the VHH antibody domain in (D).

[0391]

[0121] A compound according to any one of items

[23] to

[0119] , in which the binding affinity (KD value) of the VHH antibody domain in (D) to human NKp30 is not more than two times stronger than the binding affinity (KD value) of a VHH antibody domain consisting of a sequence from the table of VHH sequences having the highest sequence identity to the sequence of the VHH antibody domain in (D).

[0392]

[0122] A compound according to any one of items

[23] to

[0119] , in which the binding affinity (KD value) of the VHH antibody domain in (D) to human NKp30 is not more than 1.5 times stronger than the binding affinity (KD value) of a VHH antibody domain consisting of a sequence from the table of VHH sequences having the highest sequence identity to the sequence of the VHH antibody domain in (D).

[0393]

[0123] The KD value / the k on speed / k off The rate is measured by Biolayer Interference Kinetics Measurement in KB buffer (PBS + 0.1% Tween-20 + 1% BSA) at 25 °C and 1000 rpm, the compound according to any one of items

[92] to

[0122] .

[0394]

[0124] The compound according to any one of items [1] to

[0123] , wherein the VHH antibody domain or a fragment thereof competes with human B7-H6 for binding to human NKp30.

[0395]

[0125] The compound according to any one of items [1] to

[0123] , wherein the VHH antibody domain or a fragment thereof does not compete with human B7-H6 for binding to human NKp30.

[0396]

[0126] The compound according to any one of items [1] to

[0123] , wherein the VHH antibody domain or a fragment thereof partially competes with human B7-H6 for binding to human NKp30.

[0397]

[0127] The compound according to any one of items

[23] to

[0126] , wherein the VHH antibody domain of (A) comprises any one of the VHH sequences VHH1, VHH2, VHH3, VHH4, VHH5, VHH8, VHH9, VHH10, VHH11, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the table of VHH sequences. (For compounds having such a VHH antibody domain, as can be seen from, for example, Example 3, Table 1, a KD in the range of E-09 or stronger was observed for binding to NKp30.)

[0398]

[0128] The compound according to any one of items

[23] to

[0126] , wherein the VHH antibody domain of (A) comprises any one of the VHH sequences VHH1, VHH3, VHH5, VHH8, VHH10, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the table of VHH sequences. (For compounds having such a VHH antibody domain, as can be seen from, for example, Example 3, Table 1, a KD in the range of E-10 or stronger was observed for binding to NKp30.)

[0399]

[0129] The VHH antibody domain of (A) is a compound of any one of items

[23] to

[0126] , which comprises the VHH sequence VHH15 shown in the table of VHH sequences. (For a compound having such a VHH antibody domain, as can be seen from, for example, Example 3 and Table 1, a KD in the range of E-12 or stronger and a kD in the range of E-07 or lower.) off was observed for binding to NKp30.

[0400]

[0130] The VHH antibody domain of (A) is any one of the compounds of items

[23] to

[0126] , which includes any one of the VHH sequences VHH1, VHH2, VHH3, VHH5, VHH6, VHH8, VHH9, VHH10, VHH11, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the table of VHH sequences. (For a compound having such a VHH antibody domain, as an example, Example 3, Table 1, k in the range of E-05 is used.) onwas observed for binding to NKp30.

[0401]

[0131] The VHH antibody domain of (A) is any one of the compounds of items

[23] to

[0126] , which includes any one of the VHH sequences VHH1, VHH2, VHH3, VHH4, VHH5, VHH8, VHH10, VHH11, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the table of VHH sequences. (For a compound having such a VHH antibody domain, as can be seen from, for example, Example 3 and Table 1, a k value in the range of E-04 or lower is used.) off was observed for binding to NKp30.

[0402]

[0132] The VHH antibody domain of (A) is any one of the compounds of items

[23] to

[0126] , which includes any one of the VHH sequences VHH10, VHH15, or VHH16 shown in the table of VHH sequences. (For a compound having such a VHH antibody domain, as can be seen from, for example, Example 3 and Table 1, a k value in the range of E-05 or lower is used.) off was observed for binding to NKp30.

[0403]

[0133] The compound according to any one of items

[23] to

[0126] , wherein the VHH antibody domain of (A) comprises any one of the VHH sequences VHH1, VHH3, VHH5, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the table of VHH sequences. (For a compound having such a VHH antibody domain, as can be seen from, for example, Example 3 and Table 1, competition with B7-H6 is observed. As shown in Example 4, such a VHH antibody domain results in a compound that mediates lysis of targeted cells with high efficiency.)

[0404]

[0134] A compound according to any one of items

[23] to

[0126] , wherein the VHH antibody domain of (A) comprises any one of the VHH sequences VHH1, VHH5, or VHH16 shown in the table of VHH sequences.

[0405]

[0135] The compound according to any one of items

[23] to

[0126] , wherein the VHH antibody domain of (A) comprises any one of the VHH sequences VHH2, VHH6, VHH7, VHH8, VHH9, VHH10, or VHH11 shown in the table of VHH sequences. (For compounds having such VHH antibody domains, as can be seen from, for example, Example 3, Table 1, competition with no B7-H6 is observed. As shown in Example 4, such VHH antibody domains result in compounds that mediate lysis of targeted cells, but with lower efficiency.)

[0406]

[0136] A compound according to any one of items

[23] to

[0126] , wherein the VHH antibody domain of (A) comprises any one of the VHH sequences VHH2, VHH4, or VHH8 shown in the table of VHH sequences.

[0407]

[0137] A compound according to any one of items

[23] to

[0126] , wherein the VHH antibody domain of (A) comprises any one of the VHH sequences VHH2 or VHH8 shown in the table of VHH sequences.

[0408]

[0138] The VHH antibody domain of (A) is any one of the compounds of items

[23] to

[0126] , which comprises the VHH sequence VHH4 shown in the table of VHH sequences. (For a compound having such a VHH antibody domain, partial competition with B7-H6 is observed, as can be seen from, for example, Example 3, Table 1.)

[0409]

[0139] The compound according to any one of items

[23] to

[0126] , wherein the VHH antibody domain of (A) comprises any one of the VHH sequences VHH12 or VHH15VHH shown in the sequence table. (For compounds having such a VHH antibody domain, no competition with the partial competitor VHH4 is observed, as can be seen, for example, from Example 3.)

[0410]

[0140] The VHH antibody domain of (A) is any one of the compounds of items

[23] to

[0126] , which includes any one of the VHH sequences VHH8, VHH9, VHH10, or VHH11 shown in the table of VHH sequences. (As can be seen from Example 3, for example, compounds having such a VHH antibody domain belong to a bin of a specific epitope having a common characteristic.)

[0411]

[0141] The VHH antibody domain of (A) is any one of the compounds of items

[23] to

[0126] , which includes any one of the VHH sequences VHH2 or VHH6 shown in the table of VHH sequences. (As can be seen from Example 3, for example, compounds having such a VHH antibody domain belong to a bin of a specific epitope having a common characteristic.)

[0412]

[0142] The compound according to any one of items

[23] to

[0126] , wherein the VHH antibody domain of (A) comprises any one of the VHH sequences VHH2, VHH6, or VHH7 shown in the table of VHH sequences. (As can be seen from Example 3, for example, compounds having such VHH antibody domains are unique with respect to their targeting of epitopes.)

[0413]

[0143] The VHH antibody domain or fragment thereof in (A) is any one of the compounds of items [1] to

[22] or

[61] to

[0142] , which comprises the complementarity determining regions CDR1, CDR2, and CDR3 of any one of the VHH sequences VHH1, VHH2, VHH3, VHH4, VHH5, VHH8, VHH9, VHH10, VHH11, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the CDR table. (For compounds having such VHH antibody domains, as can be seen from, for example, Example 3, Table 1, a KD in the range of E-09 or stronger was observed for binding to NKp30.)

[0414]

[0144] The VHH antibody domain or fragment thereof in (A) is a compound of any one of items [1] to

[22] or

[61] to

[0142] , which comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of any one of VHH1, VHH3, VHH5, VHH8, VHH10, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the CDR table. (For compounds having such a VHH antibody domain, as can be seen from, for example, Example 3, Table 1, a KD in the range of E-10 or stronger was observed for binding to NKp30.)

[0415]

[0145] The VHH antibody domain or fragment thereof in (A) is any one of the compounds of items [1] to

[22] or

[61] to

[0142] , which includes one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH15 shown in the CDR table. (For a compound having such a VHH antibody domain, as can be seen from, for example, Example 3 and Table 1, a KD in the range of E-12 or stronger and a kD in the range of E-07 or lower.) off was observed for binding to NKp30.

[0416]

[0146] The VHH antibody domain or fragment thereof in (A) is any one of the compounds of items [1] to

[22] or

[61] to

[0142] , which includes one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH1, VHH2, VHH3, VHH5, VHH6, VHH8, VHH9, VHH10, VHH11, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the CDR table. (For a compound having such a VHH antibody domain, for example, as can be seen from Example 3, Table 1, k in the range of E-05 is used.) on was observed for binding to NKp30.

[0417]

[0147] The VHH antibody domain or fragment thereof in (A) is any one of the compounds of items [1] to

[22] or

[61] to

[0142] , which includes one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH1, VHH2, VHH3, VHH4, VHH5, VHH8, VHH10, VHH11, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the CDR table. (For a compound having such a VHH antibody domain, as can be seen from, for example, Example 3, Table 1, a k value in the range of E-04 or lower is used.) off was observed for binding to NKp30.

[0418]

[0148] The VHH antibody domain or fragment thereof in (A) is any one of the compounds of items [1] to

[22] or

[61] to

[0142] , which comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH10, VHH15, or VHH16 shown in the CDR table. (For a compound having such a VHH antibody domain, as can be seen from, for example, Example 3, Table 1, k in the range of E-05 or lower. off was observed for binding to NKp30.

[0419]

[0149] The VHH antibody domain or fragment thereof in (A) is any one of the compounds of items [1] to

[22] or

[61] to

[0142] , which comprises the complementarity determining regions CDR1, CDR2, and CDR3 of one of VHH1, VHH3, VHH5, VHH12, VHH13, VHH14, VHH15, or VHH16 shown in the CDR table. (For a compound having such a VHH antibody domain, as can be seen from, for example, Example 3, Table 1, competition with B7-H6 is observed. As shown in Example 4, such a VHH antibody domain results in a compound that mediates lysis of targeted cells with high efficiency.)

[0420]

[0150] The VHH antibody domain or fragment thereof in (A) is any one of the compounds of items [1] to

[22] or

[61] to

[0142] , comprising the complementarity determining regions CDR1, CDR2, and CDR3 of one of VHH1, VHH5, or VHH16 shown in the CDR table.

[0421]

[0151] The VHH antibody domain or fragment thereof in (A) is any one of the compounds of items [1] to

[22] or

[61] to

[0142] , which comprises the complementarity determining regions CDR1, CDR2, and CDR3 of one of VHH2, VHH6, VHH7, VHH8, VHH9, VHH10, or VHH11 as shown in the CDR table. (For compounds having such VHH antibody domains, as can be seen from, for example, Example 3, Table 1, no competition with B7-H6 is observed. As shown in Example 4, such VHH antibody domains result in compounds that mediate lysis of targeted cells, but with lower efficiency.)

[0422]

[0152] The VHH antibody domain or fragment thereof in (A) is any one of the compounds of items [1] to

[22] or

[61] to

[0142] , comprising the complementarity determining regions CDR1, CDR2, and CDR3 of one of VHH2, VHH4, or VHH8 shown in the CDR table.

[0423]

[0153] The VHH antibody domain or fragment thereof in (A) is any one of the compounds of items [1] to

[22] or

[61] to

[0142] , comprising one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH2 or VHH8 shown in the CDR table.

[0424]

[0154] The VHH antibody domain or fragment thereof in (A) is any one of the compounds of items [1] to

[22] or

[61] to

[0142] , which contains the complementarity determining regions CDR1, CDR2, and CDR3 of VHH4 shown in the CDR table. (As can be seen from, for example, Example 3, Table 1, partial competition with B7-H6 is observed for compounds having such a VHH antibody domain.)

[0425]

[0155] The VHH antibody domain or fragment thereof in (A) is any one of the compounds of items [1] to

[22] or

[61] to

[0142] , which comprises the complementarity determining regions CDR1, CDR2, and CDR3 of one of VHH12 or VHH15 shown in the CDR table. (For compounds having such a VHH antibody domain, no competition with the partial competitor VHH4 is observed, as can be seen from Example 3, for example.)

[0426]

[0156] The VHH antibody domain or fragment thereof in (A) is any one of the compounds of items [1] to

[22] or

[61] to

[0142] , which comprises the complementarity determining regions CDR1, CDR2, and CDR3 of one of VHH8, VHH9, VHH10, or VHH11 shown in the CDR table. (As can be seen from Example 3, for example, compounds having such a VHH antibody domain belong to a bin of a specific epitope having a common characteristic.)

[0427]

[0157] The VHH antibody domain or fragment thereof in (A) is any one of the compounds of items [1] to

[22] or

[61] to

[0142] , which comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH2 or VHH6 shown in the CDR table. (As can be seen from Example 3, for example, compounds having such a VHH antibody domain belong to a bin of a specific epitope having a common characteristic.)

[0428]

[0158] The VHH antibody domain or fragment thereof in (A) is a compound according to any one of items [1] to

[22] or

[61] to

[0142] , which comprises the complementarity determining regions CDR1, CDR2, and CDR3 of one of VHH2, VHH6, or VHH7 shown in the CDR table. (As can be seen from Example 3, for example, compounds having such VHH antibody domains are unique with respect to their targeting of epitopes.)

[0429]

[0159] The compound according to any one of items [1] to

[0158] , wherein the compound is a molecule.

[0430]

[0160] The compound according to any one of items [1] to

[0159] , wherein the compound includes or is a protein.

[0431]

[0161] The compound according to any one of items [1] to

[0160] , wherein the compound is a protein.

[0432]

[0162] The compound is a compound according to any one of items

[23] to

[0161] , comprising a VHH antibody domain or a fragment thereof according to (A), (B), or (C).

[0433]

[0163] The compound is a compound according to any one of items

[23] to

[0161] , comprising a VHH antibody domain or a fragment thereof according to (A), (B), or (D).

[0434]

[0164] The compound is a compound according to any one of items

[23] to

[0161] , comprising a VHH antibody domain or a fragment thereof according to (A), (C), or (D).

[0435]

[0165] The compound is a compound according to any one of items

[23] to

[0161] , comprising a VHH antibody domain or a fragment thereof according to (A) or (B).

[0436]

[0166] The compound is a compound according to any one of items

[23] to

[0161] , comprising a VHH antibody domain or a fragment thereof according to (A) or (C).

[0437]

[0167] The compound is a compound according to any one of items

[23] to

[0161] , comprising a VHH antibody domain or a fragment thereof according to (A) or (D).

[0438]

[0168] The compound is a compound according to any one of items

[23] to

[0161] , comprising a VHH antibody domain according to (A) or a fragment thereof.

[0439]

[0169] The compound is a compound according to any one of items

[23] to

[0161] , comprising a VHH antibody domain or a fragment thereof according to (B).

[0440]

[0170] A compound according to any one of items [1] to

[22] or

[61] to

[0169] , wherein the VHH antibody domain or fragment thereof comprises a complementarity determining region according to (a) or (b).

[0441]

[0171] A compound according to any one of items [1] to

[22] or

[61] to

[0169] , wherein the VHH antibody domain or fragment thereof comprises a complementarity determining region according to (a) or (c).

[0442]

[0172] A compound according to any one of items [1] to

[22] or

[61] to

[0169] , wherein the VHH antibody domain or fragment thereof comprises a complementarity determining region according to (a).

[0443]

[0173] A compound according to any one of items [1] to

[22] or

[61] to

[0169] , wherein the VHH antibody domain or fragment thereof comprises a complementarity determining region according to (b).

[0444]

[0174] The compound according to any one of items [1] to

[0173] , further comprising a targeting moiety.

[0445]

[0175] A compound according to any one of items [1] to

[0174] , wherein all components of the compound are covalently linked.

[0446]

[0176] A compound according to any one of items

[0174] or

[0175] , wherein the targeting moiety is a group of molecules that specifically binds to a target molecule or a fragment thereof.

[0447]

[0177] The compound according to item

[0176] , wherein the target molecule is a receptor on the surface of a cell.

[0448]

[0178] The compound according to any one of items

[0176] or

[0177] , wherein the target molecule is an antigen present on the surface of a target cell.

[0449]

[0179] A compound according to any one of items

[0174] to

[0178] , wherein the targeting moiety is a protein, peptide, peptidomimetic, nucleic acid, oligonucleotide, or small molecule.

[0450]

[0180] The compound according to any one of items

[0174] to

[0179] , wherein the targeting moiety comprises a protein or is a protein.

[0451]

[0181] The compound according to any one of items

[0174] to

[0180] , wherein the targeting moiety comprises a protein.

[0452]

[0182] The compound according to any one of items

[0174] to

[0181] , wherein the targeting component is a protein.

[0453]

[0183] The compound according to any one of items

[0174] to

[0182] , wherein the targeting moiety is a protein ligand that specifically binds to a receptor on the surface of a cell.

[0454]

[0184] The compound according to any one of items

[0174] to

[0182] , wherein the targeting component is an antibody or an antigen-binding fragment thereof.

[0455]

[0185] A compound according to any one of items

[0174] to

[0184] , wherein the targeting moiety is capable of specifically binding to a tumor-associated antigen.

[0456]

[0186] The compound according to any one of items [1] to

[0185] , wherein the compound is a bispecific antibody.

[0457]

[0187] The compound according to any one of items [1] to

[0186] , wherein the compound is a bispecific antibody prepared by SEED (strand exchange engineered domain) technology.

[0458]

[0188] A compound according to any one of items

[0186] or

[0187] , wherein one binding site of the bispecific antibody is formed by the VHH antibody domain or a fragment thereof, and one binding site of the bispecific antibody is formed by the targeting moiety.

[0459]

[0189] The compound according to any one of items

[0184] to

[0188] , wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, Fv, scFv, diabody, and VHH.

[0460]

[0190] The compound according to any one of items

[0184] to

[0188] , wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, and Fv.

[0461]

[0191] The compound according to any one of items

[0184] to

[0188] , wherein the antigen-binding fragment is a Fab.

[0462]

[0192] The compound according to any one of items

[0184] to

[0188] , wherein the antigen-binding fragment is selected from the group consisting of scFv, diabody, and VHH.

[0463]

[0193] A compound according to any one of items

[0184] to

[0192] , wherein the antigen-binding fragment is an antigen-binding fragment of an antibody having a SEED (strand exchange engineered domain) format.

[0464]

[0194] A compound according to any one of items

[0174] to

[0193] , wherein the targeting moiety is capable of specifically binding to an antigen present on the surface of a target cell.

[0465]

[0195] The compound according to any one of items

[0184] to

[0194] , wherein the antibody is an antibody against an antigen present on the surface of a target cell.

[0466]

[0196] A compound according to any one of items

[0184] to

[0195] , wherein the antigen-binding fragment is an antigen-binding fragment of an antibody against an antigen present on the surface of a target cell.

[0467]

[0197] A compound according to any one of items

[0194] to

[0196] , wherein the binding of the targeting moiety to the antigen present on the surface of the target cell enables the compound to be specifically recruited to the target cell.

[0468]

[0198] The compound according to any one of items

[0178] to

[0197] , wherein the antigen present on the surface of the target cell is a tumor-associated antigen.

[0469]

[0199] A compound according to any one of items

[0174] to

[0198] , wherein the targeting moiety is capable of specifically binding to a tumor-associated antigen.

[0470]

[0200] The compound according to any one of items

[0185] to

[0199] , wherein the tumor-associated antigen is an antigen present on the surface of a tumor cell.

[0471]

[0201] The compound according to any one of items

[0185] to

[0199] , wherein the tumor-associated antigen is EGFR (epidermal growth factor receptor).

[0472]

[0202] The compound according to any one of items [1] to

[0201] , wherein the compound is a bispecific or multispecific molecule.

[0473]

[0203] The compound according to any one of items [1] to

[0202] , wherein the compound comprises an antibody Fc region.

[0474]

[0204] The compound according to any one of items [1] to

[0203] , wherein the compound comprises an antibody Fc region capable of binding to an Fc receptor.

[0475]

[0205] The compound according to any one of items [1] to

[0204] , comprising an antibody Fc region that is incapable of binding to an Fc receptor.

[0476]

[0206] The compound according to any one of items [1] to

[0205] , wherein the compound does not contain an antibody Fc region having effector capability.

[0477]

[0207] The compound according to any one of items [1] to

[0205] , wherein the compound comprises an antibody Fc region having effector capability.

[0478]

[0208] The compound according to any one of items [1] to

[0207] , which does not contain an antibody Fc region capable of inducing ADCC (antibody-dependent cellular cytotoxicity).

[0479]

[0209] The compound according to any one of items [1] to

[0207] , which comprises an antibody Fc region capable of inducing ADCC.

[0480]

[0210] The compound according to any one of items [1] to

[0209] , wherein the compound comprises an antibody Fc region that is not capable of inducing ADCC.

[0481]

[0211] A compound according to any one of items [1] to

[0210] , which is capable of inducing both FcγRIIIa signaling and positive (i.e., activating NK cells) NKp30 signaling.

[0482]

[0212] The compound according to any one of items [1] to

[0211] , wherein the compound is capable of specifically binding to NKp30 on NK cells.

[0483]

[0213] The compound according to any one of items [1] to

[0212] , wherein the compound is capable of activating an NK cell by binding to NKp30 on the NK cell.

[0484]

[0214] A compound according to any one of items

[0243] to

[0213] , wherein binding of the compound to NKp30 on an NK cell activates the NK cell.

[0485]

[0215] The compound according to any one of items [1] to

[0214] , which is an agonist of NKp30.

[0486]

[0216] A compound according to any one of items [1] to

[0215] , wherein the compound is capable of activating NK cells when the VHH antibody domain or a fragment thereof binds to NKp30 on an NK cell.

[0487]

[0217] The compound according to any one of items [1] to

[0216] , wherein binding of the compound to NKp30 on an NK cell activates the NK cell.

[0488]

[0218] A compound according to any one of items

[0211] to

[0217] , wherein the activation of NK cells is measured by measuring the expression of the activation marker CD69 by flow cytometry.

[0489] The compound is 51 A compound according to any one of items [1] to

[0218] , which exhibits cytotoxic activity in a Cr release assay.

[0490]

[0220] Applicable 51 Cr release assay was performed for 4 h. 51A Cr release assay was performed in which human PBMCs were used as effector cells at an effector to target cell (E:T) ratio of 80:1, and a higher percent lysis indicates improved cytotoxic activity of the compound according to item

[0219] .

[0491]

[0221] Applicable 51 Cr release assays are performed as described in Repp et al., 2011. Compounds according to any one of items

[0219] to

[0220] .

[0492]

[0222] The compound according to any one of items [1] to

[0221] , wherein binding of the compound to NKp30 on NK cells results in the release of interferon-γ (IFN-γ).

[0493]

[0223] The release of IFN-γ is measured as follows: isolated human NK cells are incubated overnight in medium containing 100 U / ml recombinant human interleukin-2, A431 cells in different wells are seeded and incubated for 3 h, compounds are added to a final concentration of 85 nM, followed by addition of NK cells at an E:T ratio of 5:1, and human IFN-γ is analyzed by ELISA in the culture supernatants after 24 h, compounds according to item

[0222] .

[0494]

[0224] The compound according to any one of items [1] to

[0223] , wherein binding of the compound to NKp30 on NK cells results in the release of tumor necrosis factor-α (TNF-α).

[0495]

[0225] The release of TNF-α is measured as follows: isolated human NK cells are incubated overnight in medium containing 100 U / ml recombinant human interleukin-2, A431 cells in different wells are seeded and incubated for 3 h, compounds are added to a final concentration of 85 nM, followed by addition of NK cells at an E:T ratio of 5:1, and human IFN-γ is analyzed by ELISA in the culture supernatants after 24 h, compounds according to item

[0224] .

[0496]

[0226] A pharmaceutical composition comprising a compound according to any one of items [1] to

[0225] .

[0497]

[0227] The pharmaceutical composition according to item

[0226] , wherein the pharmaceutical composition comprises a pharma- ceutically acceptable carrier, diluent, and / or excipient.

[0498]

[0228] The pharmaceutical composition according to any one of items

[0226] to

[0227] , further comprising at least one additional adjuvant, antioxidant, buffer, bulking agent, colorant, emulsifier, filler, flavoring agent, preservative, stabilizer, suspending agent, and / or other conventional pharmaceutical auxiliary.

[0499]

[0229] A compound according to any one of items [1] to

[0225] or a pharmaceutical composition according to any one of items

[0226] to

[0228] for use as a medicine.

[0500]

[0230] A compound according to any one of items [1] to

[0225] or a pharmaceutical composition according to any one of items

[0226] to

[0228] for use in the treatment of cancer.

[0501]

[0231] A compound according to any one of items [1] to

[0225] or a pharmaceutical composition according to any one of items

[0226] to

[0228] for use in treating a malignant tumor.

[0502]

[0232] A compound according to any one of items [1] to

[0225] or a pharmaceutical composition according to any one of items

[0226] to

[0228] , wherein the compound / pharmaceutical composition is for use in treating humans.

[0503]

[0233] A method for treating a disease in a patient in need thereof, comprising the step of administering to the patient a therapeutically effective amount of a compound according to any one of items [1] to

[0225] or a pharmaceutical composition according to any one of items

[0226] to

[0228] .

[0504]

[0234] The method according to item

[0233] , wherein the disease is cancer.

[0505]

[0235] The method according to item

[0233] , wherein the disease is a malignant tumor.

[0506]

[0236] The method according to any one of items

[0233] to

[0235] , wherein the patient is a human.

[0507]

[0237] Use of a compound according to any one of items [1] to

[0225] or a pharmaceutical composition according to any one of items

[0226] to

[0228] for the manufacture of a medicament.

[0508]

[0238] Use of a compound according to any one of items [1] to

[0225] or a pharmaceutical composition according to any one of items

[0226] to

[0228] for the manufacture of a medicament for the treatment of cancer.

[0509]

[0239] Use of a compound according to any one of items [1] to

[0225] or a pharmaceutical composition according to any one of items

[0226] to

[0228] for the manufacture of a medicament for the treatment of a malignant tumor.

[0510]

[0240] The use according to any one of items

[0237] to

[0239] , wherein the medicament is prepared for administration to a human.

[0511]

[0241] A compound or pharmaceutical composition for use according to any one of items

[0230] or

[0231] , or a method according to any one of items

[0234] or

[0235] , or for use according to any one of items

[0238] or

[0239] , wherein the cancer or malignant tumor is a human disease.

[0512]

[0242] The compound comprises a sequence represented by SEQ ID NO:1 (or a fragment thereof) and has one or more amino acid substitutions compared to the sequence of SEQ ID NO:1, wherein if such amino acid substitutions are located outside the CDRs, the amino acid substitution(s) is / are selected from the following: D1E, Q5V, L11V, T24A, E44G, H45L, G47W, F70I, V79L, K87R, P88A, Q116L, or any one of the compounds of items [1] to

[22] or

[85] to

[0110] or

[0123] to

[0126] or

[0143] to

[0161] or

[0170] to

[0225] or

[0232] ;

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0513] The compound comprises a sequence represented by SEQ ID NO:1 (or a fragment thereof) having at least the following amino acid substitutions: D1E, Q5V, L11V, V79L, K87R, P88A, Q116L; and optionally one or more further amino acid substitutions compared to the sequence of SEQ ID NO:1, where if such amino acid substitutions are located outside the CDRs, such further amino acid substitution(s) are selected from the following: A compound selected from T24A, E44G, H45L, F70I, or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0514]

[0244] The compound comprises a sequence represented by SEQ ID NO:1 (or a fragment thereof) with the additional modification (b) according to any one of items [1] to

[11] , or with the additional modification (c) according to any one of items [1] or

[12] to

[22] , or

[85] to

[0110] , or

[0123] to

[0126] , or

[0143] to

[0161] , or

[0170] to

[0225] , or

[0226] to

[0228] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0515]

[0245] The compound comprises a sequence represented by SEQ ID NO:1 (or a fragment thereof) with an additional modification (b) according to any one of items [1] to

[11] , or with an additional modification (c) according to any one of items [1] or

[12] to

[22] ; and with one or more further amino acid substitutions compared to the sequence of SEQ ID NO:1, wherein the amino acid substitution(s) are selected from the following: D1E, Q5V, L11V, T24A, E44G, H45L, G47W, F70I, V79L, K87R, P88A, Q116L, or a compound of any one of items [1] to

[22] or

[85] to

[0110] , or

[0123] to

[0126] , or

[0143] to

[0161] , or

[0170] to

[0225] , or

[026] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0516] The compound comprises a sequence represented by SEQ ID NO:1 (or a fragment thereof) with an additional modification (b) according to any one of items [1] to

[11] , or with an additional modification (c) according to any one of items [1] or

[12] to

[22] ; and with the amino acid substitutions D1E, Q5V, L11V, V79L, K87R, P88A, Q116L; and optionally with one or more further amino acid substitutions compared to the sequence of SEQ ID NO:1, wherein the further amino acid substitution(s) are as follows: A compound selected from T24A, E44G, H45L, F70I, or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0517] The compound has the following SEQ ID NO: A compound according to any one of items [1] to

[22] or

[85] to

[0110] or

[0123] to

[0126] or

[0143] to

[0161] or

[0170] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0518]

[0248] The compound is a compound of any one of items [1] to

[22] , or

[85] to

[0110] , or

[0123] to

[0126] , or

[0143] to

[0161] , or

[0170] to

[0225] , or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] , or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] , or

[0241] , or a method according to any one of items

[0233] to

[0236] , or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0519] The compound has the following SEQ ID NO: A compound according to any one of items [1] to

[22] or

[85] to

[0110] or

[0123] to

[0126] or

[0143] to

[0161] or

[0170] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0520]

[0250] The compound is a compound of any one of items [1] to

[22] , or

[85] to

[0110] , or

[0123] to

[0126] , or

[0143] to

[0161] , or

[0170] to

[0225] , or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] , or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] , or

[0241] , or a method according to any one of items

[0233] to

[0236] , or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0521] The compound has the following SEQ ID NO: A compound according to any one of items [1] to

[22] or

[85] to

[0110] or

[0123] to

[0126] or

[0143] to

[0161] or

[0170] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0522]

[0252] The compound is a compound of any one of items [1] to

[22] or

[85] to

[0110] or

[0123] to

[0126] or

[0143] to

[0161] or

[0170] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0523]

[0253] The compound is any one of the compounds of items [1] to

[22] or

[85] to

[0110] or

[0123] to

[0126] or

[0143] to

[0161] or

[0170] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0524]

[0254] The compound is a compound of any one of items [1] to

[22] , or

[85] to

[0110] , or

[0123] to

[0126] , or

[0143] to

[0161] , or

[0170] to

[0225] , or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] , or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] , or

[0241] , or a method according to any one of items

[0233] to

[0236] , or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0525]

[0255] The compound includes a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or a fragment thereof; or an additional modification according to any one of items [1] to

[11] (b) or an additional modification according to any one of items [1] or

[12] to

[22] (c), A compound according to any one of items

[0161] or

[0170] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0526]

[0256] The compound comprises a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or a fragment thereof; with the additional modification (b) according to any one of items [1] to

[11] , or with the additional modification (c) according to any one of items [1] or

[12] to

[22] , or

[85] to

[0110] , or

[0123] to

[0126] , or

[0143] to

[0161] , or

[0170] to

[022] .

[0235] or

[0236] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0527] The compound includes a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, or a fragment thereof; with the additional modification (b) according to any one of items [1] to

[11] , or with the additional modification (c) according to any one of items [1] or

[12] to

[22] , or items [1] to

[22] or

[85] to

[0110] or

[0123] to

[0126] or [014

[0237] to

[0238] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0528]

[0258] The compound includes a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 77, or a fragment thereof; with the additional modification (b) according to any one of items [1] to

[11] , or with the additional modification (c) according to any one of items [1] or

[12] to

[22] , or

[85] to

[0110] , or

[0123] to

[0126] , or

[0143] to

[0161] , or

[0170] to [

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0529] The compound includes a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, or a fragment thereof; with the additional modification (b) according to any one of items [1] to

[11] , or with the additional modification (c) according to any one of items [1] or

[12] to

[22] , or items [1] to

[22] or

[85] to

[0110] or

[0123] to

[0126] or [014

[0237] to

[0238] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0530]

[0260] The compound includes a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 78, or a fragment thereof; with the additional modification (b) according to any one of items [1] to

[11] , or with the additional modification (c) according to any one of items [1] or

[12] to

[22] , or with the additional modification (c) according to any one of items [1] or

[12] to

[22] , or with the additional modification (c) according to any one of items [1] or

[12] to

[22] ,

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0531]

[0261] The compound comprises a sequence as defined by SEQ ID NO: 77 or a fragment thereof, and has an additional modification (b) according to any one of items [1] to

[11] , or an additional modification (c) according to any one of items [1] or

[12] to

[22] , or

[85] to

[0110] , or

[0123] to

[0126] , or

[0143] to

[0161] , or

[0170] to

[0225] , or

[0226] to

[0228] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0532]

[0262] The compound comprises a sequence as defined by SEQ ID NO: 78 or a fragment thereof, and has an additional modification (b) according to any one of items [1] to

[11] , or an additional modification (c) according to any one of items [1] or

[12] to

[22] , or

[85] to

[0110] , or

[0123] to

[0126] , or

[0143] to

[0161] , or

[0170] to

[0225] , or

[0226] to

[0228] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0533]

[0263] The compound comprises a sequence represented by SEQ ID NO:2 (or a fragment thereof) and has one or more amino acid substitutions compared to the sequence of SEQ ID NO:2, wherein if such amino acid substitutions are located outside the CDRs, the amino acid substitution(s) is / are selected from the following: D1E, Q5V, L11V, A14P, V24A, F37V, A44G, R45L, S47W, V79L, K87R, P88A, Q118L, or any one of the compounds of items [1] to

[22] , or

[85] to

[0110] , or

[0123] to

[0126] , or

[0143] to

[0161] , or

[0170] to

[0225] , or

[0232] . A pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0534] The compound comprises the sequence represented by SEQ ID NO:2 (or a fragment thereof) having the amino acid substitutions D1E, Q5V, L11V, K87R, P88A, Q118L; and optionally one or more further amino acid substitutions compared to the sequence of SEQ ID NO:2, where if such amino acid substitutions are located outside the CDRs, then such further amino acid substitution(s) are selected from the following: A compound selected from A14P, V24A, A44G, R45L, S47W, V79L, or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0535] The compound comprises the sequence represented by SEQ ID NO:2 (or a fragment thereof) with the amino acid substitutions D1E, Q5V, L11V, A14P, V24A, K87R, P88A, Q118L; and optionally with one or more further amino acid substitutions compared to the sequence of SEQ ID NO:2, where if such amino acid substitutions are located outside the CDRs, then such further amino acid substitution(s) are selected from the following: A compound selected from A44G, R45L, S47W, V79L, or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0536]

[0266] The compound comprises a sequence represented by SEQ ID NO:2 (or a fragment thereof) with the additional modification (b) according to any one of items [1] to

[11] , or with the additional modification (c) according to any one of items [1] or

[12] to

[22] , or

[85] to

[0110] , or

[0123] to

[0126] , or

[0143] to

[0161] , or

[0170] to

[0225] , or

[0226] to

[0228] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0537] The compound comprises a sequence represented by SEQ ID NO:2 (or a fragment thereof) with an additional modification (b) according to any one of items [1] to

[11] , or with an additional modification (c) according to any one of items [1] or

[12] to

[22] ; and with one or more further amino acid substitutions compared to the sequence of SEQ ID NO:2, wherein the amino acid substitution(s) is / are selected from the following: D1E, Q5V, L11V, A14P, V24A, F37V, A44G, R45L, S47W, V79L, K87R, P88A, Q118L, or a compound selected from any one of items [1] to

[22] or

[85] to

[0110] , or

[0123] to

[0126] , or

[0143] to

[0161] , or

[0170] to

[0225] , or A pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0538] The compound comprises a sequence represented by SEQ ID NO:2 (or a fragment thereof) with an additional modification (b) according to any one of items [1] to

[11] , or with an additional modification (c) according to any one of items [1] or

[12] to

[22] ; and with the amino acid substitutions D1E, Q5V, L11V, K87R, P88A, Q118L; and optionally with one or more further amino acid substitutions compared to the sequence of SEQ ID NO:2, wherein the further amino acid substitution(s) are as follows: A compound selected from A14P, V24A, A44G, R45L, S47W, V79L, or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0539] The compound comprises a sequence represented by SEQ ID NO:2 (or a fragment thereof) with an additional modification (b) according to any one of items [1] to

[11] , or with an additional modification (c) according to any one of items [1] or

[12] to

[22] ; and with the amino acid substitutions D1E, Q5V, L11V, A14P, V24A, K87R, P88A, Q118L; and optionally with one or more further amino acid substitutions compared to the sequence of SEQ ID NO:2, wherein the further amino acid substitution(s) are as follows: A compound selected from A44G, R45L, S47W, V79L, or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0540] The compound has the following SEQ ID NO: A compound according to any one of items [1] to

[22] or

[85] to

[0110] or

[0123] to

[0126] or

[0143] to

[0161] or

[0170] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0541]

[0271] The compound is a compound of any one of items [1] to

[22] or

[85] to

[0110] or

[0123] to

[0126] or

[0143] to

[0161] or

[0170] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0542] The compound includes a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:94, or a fragment thereof; or an additional modification according to (b) in accordance with any one of items [1] to

[11] , or an additional modification according to (c) in accordance with any one of items [1] or

[12] to [2 ... to

[22] or

[85] to

[0110] or

[0123] to

[0126] or A compound according to any one of items

[0143] to

[0161] or

[0170] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0543] The compound includes a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:94, or a fragment thereof; or includes any one of items [1] to

[22] or

[85] to

[0110] or

[0123] to

[0126] or

[0143] to

[0161] , with the additional modification of (b) according to any one of items [1] to

[11] , or with the additional modification of (c) according to any one of items [1] or

[12] to

[22] . or a compound or pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0544] The VHH antibody domain comprises a VHH sequence VHH1 as shown in the Table of VHH Sequences or a fragment thereof, with an additional modification of (B) or (C), wherein said modification of (B) or (C) consists of one or more amino acid substitutions compared to the sequence of SEQ ID NO:1, wherein the amino acid substitution(s) is / are: A compound selected from D1E, Q5V, L11V, T24A, E44G, H45L, G47W, F70I, V79L, K87R, P88A, Q116L, or a pharmaceutical composition or compound for use according to any one of items

[0226] to

[0228] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0545] The VHH antibody domain comprises a VHH sequence VHH1 as shown in the Table of VHH Sequences or a fragment thereof, with the additional modification (B) or (C) consisting of the amino acid substitutions D1E, Q5V, L11V, V79L, K87R, P88A, Q116L compared to the sequence of SEQ ID NO:1; and optionally one or more further amino acid substitutions compared to the sequence of SEQ ID NO:1, wherein the further amino acid substitution(s) are as follows: A compound selected from T24A, E44G, H45L, F70I, or a pharmaceutical composition or compound for use according to any one of items

[0226] to

[0228] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0546]

[0276] The VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or a fragment thereof, comprising a compound of any one of items

[23] to

[45] or

[52] to

[0142] or

[0159] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0547]

[0277] The VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or a fragment thereof, comprising a compound of any one of items

[23] to

[45] or

[52] to

[0142] or

[0159] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0548]

[0278] The VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, or a fragment thereof, comprising a compound of any one of items

[23] to

[45] or

[52] to

[0142] or

[0159] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0549]

[0279] The VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:77, or a fragment thereof, comprising a compound of any one of items

[23] to

[45] or

[52] to

[0142] or

[0159] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0550]

[0280] The VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, or a fragment thereof, or a compound or pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0551]

[0281] The VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 78, or a fragment thereof, comprising a compound of any one of items

[23] to

[45] or

[52] to

[0142] or

[0159] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0552] The VHH antibody domain comprises a VHH sequence VHH1 as shown in the Table of VHH Sequences or a fragment thereof, with the additional modification (B) or (C), wherein said modification (B) or (C) consists of one or more amino acid substitutions compared to the sequence of SEQ ID NO:2, wherein the amino acid substitution(s) is / are: A compound selected from among D1E, Q5V, L11V, A14P, V24A, F37V, A44G, R45L, S47W, V79L, K87R, P88A, Q118L, or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0553] The VHH antibody domain comprises a VHH sequence VHH1 as shown in the Table of VHH Sequences or a fragment thereof, with the additional modification (B) or (C), wherein the modification (B) or (C) consists of the amino acid substitutions D1E, Q5V, L11V, K87R, P88A, Q118L compared to the sequence of SEQ ID NO:2; and optionally one or more further amino acid substitutions compared to the sequence of SEQ ID NO:2, wherein the further amino acid substitution(s) are as follows: A compound selected from A14P, V24A, A44G, R45L, S47W, V79L, or a pharmaceutical composition for use according to any one of items

[23] to

[45] or

[52] to

[0142] or

[0159] to

[0225] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0282] or

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0554] The VHH antibody domain comprises a VHH sequence VHH1 as shown in the Table of VHH Sequences or a fragment thereof, with the additional modification (B) or (C), wherein the modification (B) or (C) consists of the amino acid substitutions D1E, Q5V, L11V, A14P, V24A, K87R, P88A, Q118L compared to the sequence of SEQ ID NO:2; and optionally one or more further amino acid substitutions compared to the sequence of SEQ ID NO:2, wherein the further amino acid substitution(s) are as follows: A compound selected from A44G, R45L, S47W, V79L, or a pharmaceutical composition for use according to any one of items

[23] to

[45] or

[52] to

[0142] or

[0159] to

[0225] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0282] to

[0283] or items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0555]

[0285] The VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:94, or a fragment thereof, comprising a compound of any one of items

[23] to

[45] or

[52] to

[0142] or

[0159] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0556]

[0286] The VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:94, or a fragment thereof, comprising a compound of any one of items

[23] to

[45] or

[52] to

[0142] or

[0159] to

[0225] or

[0232] , or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] , or a method according to any one of items

[0233] to

[0236] or

[0241] , or a use according to any one of items

[0237] to

[0241] .

[0557] The compounds exhibit a binding affinity of at least about 1×10 in a BLI (BioLayer Interferometry) binding assay with the recombinant extracellular domain of NKp30 (NKp30-ECD, SEQ ID NO: 66) in KB buffer (PBS+0.1% Tween-20+1% BSA). 6 M -1 or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] or

[0242] to

[0286] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] to

[0286] , or a method according to any one of items

[0233] to

[0236] or

[0241] to

[0286] , or a use according to any one of items

[0237] to

[0286] .

[0558] The compound exhibits a binding affinity of at least about 5×10 in a BLI (BioLayer Interferometry) binding assay with the recombinant extracellular domain of NKp30 (NKp30-ECD, SEQ ID NO: 66) in KB buffer (PBS+0.1% Tween-20+1% BSA). 6 M-1 or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] or

[0242] to

[0286] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] to

[0286] , or a method according to any one of items

[0233] to

[0236] or

[0241] to

[0286] , or a use according to any one of items

[0237] to

[0286] .

[0559] The compounds exhibit a binding affinity of at least about 1×10 in a BLI (BioLayer Interferometry) binding assay with recombinant extracellular domain of NKp30 (NKp30-ECD, SEQ ID NO: 66) in KB buffer (PBS+0.1% Tween-20+1% BSA). 7 M -1 or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] or

[0242] to

[0286] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] to

[0286] , or a method according to any one of items

[0233] to

[0236] or

[0241] to

[0286] , or a use according to any one of items

[0237] to

[0286] .

[0560] The compounds exhibit a binding affinity of at least about 5×10 in a BLI (BioLayer Interferometry) binding assay with the recombinant extracellular domain of NKp30 (NKp30-ECD, SEQ ID NO: 66) in KB buffer (PBS+0.1% Tween-20+1% BSA). 7 M -1or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] or

[0242] to

[0286] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] to

[0286] , or a method according to any one of items

[0233] to

[0236] or

[0241] to

[0286] , or a use according to any one of items

[0237] to

[0286] .

[0561] The compounds exhibit a binding affinity of at least about 1×10 in a BLI (BioLayer Interferometry) binding assay with recombinant extracellular domain of NKp30 (NKp30-ECD, SEQ ID NO: 66) in KB buffer (PBS+0.1% Tween-20+1% BSA). 8 M -1 or a pharmaceutical composition for use according to any one of items

[0226] to

[0228] or

[0232] or

[0242] to

[0286] , or a compound or pharmaceutical composition for use according to any one of items

[0229] to

[0231] or

[0241] to

[0286] , or a method according to any one of items

[0233] to

[0236] or

[0241] to

[0286] , or a use according to any one of items

[0237] to

[0286] .

[0562] example The following examples describe the preparation and characterization of VHH-based NKp30 binders as disclosed in the present disclosure, and related compounds and methods, with comparative disclosure.It is understood that the various aspects of the present disclosure reflected in the examples can be practiced given the general description provided above.Although the above invention has been described in some detail by way of illustration and example for clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the present invention.

[0563] Example 1 Immunization of camelids Three camelids, one llama (Lama glama), one alpaca (Vicugna pacos), and one varizo (Lama glama x Vicugna pacos), were immunized with recombinant human (rh) NKp30 extracellular domain (ECD; produced in-house; see SEQ ID NO: 66) at preclinics GmbH, Germany. All procedures and animal care were in accordance with local animal welfare protection laws and regulations. Briefly, 200 μg rh NKp30 diluted in 1 ml PBS was emulsified in either 1 ml Freund's complete adjuvant (first immunization) or Freund's incomplete adjuvant (subsequent immunizations). Administration was performed at three sites subcutaneously. A total of six immunizations (d0, d28, d42, d56, d70, and d84) were performed over a period of 84 days. A volume of 100 ml of blood was collected on day 88, total RNA was extracted and cDNA was synthesized. All animals in this study were provided by preclinics GmbH and survived after completion of the immunization procedure.

[0564] Yeast strains and media For yeast surface display, Saccharomyces cerevisiae strain EBY100 (MATa URA3-52 trp1 leu2Δ1 his3Δ200 pep4::HIS3 prb1Δ1.6R can1 GAL(pIU211:URA3)) (Thermo Fisher Scientific) was utilized. Cells were grown in YPD medium composed of 20 g / L peptone, 20 g / L dextrose, and 10 g / L yeast extract supplemented with 10 ml / L penstrep (Gibco). Cells harboring the library plasmid (pDisp) after homologous recombination-based cloning were grown in medium supplemented with 5.4 g / L Na2HPO4 and 8.6 g / L NaH2PO4 × H2O using minimal SD bases (Clontech) together with a commercial dropout mix (Clontech) composed of all essential amino acids except tryptophan (-Trp) according to the manufacturer's instructions. For antibody induction, gene-expressing cells were transferred into SG dropout medium (-Trp) in which glucose was replaced by galactose-containing SG base (Clontech) supplemented with 10% (w / v) polyethylene glycol 800 (PEG 8000).

[0565] Plasmids for yeast surface display and library generation Homologous recombination in yeast, referred to as gap repair cloning, was exploited for the generation of VHH sublibraries. PCR amplification of VHH fragments as well as library construction were performed as described in Roth et al., 2020. Briefly, the display plasmid pDisp was digested with BsaI, followed by in-frame genetic fusion of VHH library candidates to Aga2p by replacement of the stuffer sequence resulting from gap repair cloning, ultimately allowing surface display of sdAb variants on yeast cells. Furthermore, insertion of an HA epitope linked to the C-terminus of Aga2p allowed detection of full-length VHHs on the yeast surface (Figure 1A).

[0566] Library Sorting For library sorting, rh his-tagged NKp30 ECD was purchased from Abcam. EBY100 library cells were grown overnight at 30°C and 120 rpm in SD medium with dropout mix lacking tryptophan (-Trp). For induction of surface expression, cells were transferred to SG medium with dropout mix (-Trp) for 10 min. 7 Cells / ml were transferred to the cells and followed by 48 h incubation at 20°C. Antigen binding was monitored by indirect immunofluorescence using his-tagged NKp30 followed by application of an anti-his mouse monoclonal detection antibody (SureLight® Allophycocyanin, Abcam, diluted 1:20). Full-length VHH surface expression was simultaneously detected by HA epitope tagging using a FITC-labeled rabbit polyclonal antibody (Abcam, diluted 1:20). For detection and isolation of library candidates, a BD FACSAria™ Fusion cell sorter (BD Biosciences) was employed. Control samples allowing gating of the desired cell population were employed in every experiment: untreated cells, cells incubated with labeling reagent only, or cells incubated with labeling reagent and his-tagged NKp30 or irrelevant antigen.

[0567] Antibody expression and purification After sequencing and VHH clone selection, the variants were fused to the hinge region of the SEED AG chain and cloned into pTT5, allowing the production of bispecific SEED bodies in combination with the humanized cetuximab Fab on the SEED GA chain (Figure 1A). SEED bodies were produced with wild-type IgG1 CH2 domains (effector-competent, VHH SEED bodies eff+) or in a backbone that was effector-inhibited by introducing point mutations in the Fc region that abolished immune effector functions (VHH SEED bodies eff-). For this purpose, Expi293 cells were transfected with the respective expression vectors according to the manufacturer's instructions (Thermo Fisher Scientific). After 5 days of expression, the supernatants were collected by centrifugation and purified over MabSelect antibody purification chromatography resin (GE Healthcare). Finally, the buffer was exchanged with PBS (pH 6.8) overnight using a Pur-A-Lyzer™ Maxi 3500 Dialysis Kit (Sigma Aldrich). The concentration was then determined using a Nanodrop ND-1000 (Peqlab) after sterile filtration with an Ultrafree®-CL GV 0.22 μm centrifugation device (Merck Millipore). Sample purity was assessed by determining the target monomer peak [%] via analytical size exclusion chromatography using 10 μg protein per sample on a TSKgel SuperSW3000 column (4.6×300 mm, Tosoh Bioscience LLC) on an Agilent HPLC system at a flow rate of 0.35 ml / min.

[0568] Biolayer Interference (BLI) An Octet RED96 system (ForteBio, Pall Life Science) was employed for kinetic measurements and competition assays at 25°C and 1000 rpm agitation. For binding kinetic measurements, bispecific SEED bodies were loaded onto anti-human Fc (AHC) biosensors at 5 μg / mL in PBS for 3 min, followed by rinsing the sensors with kinetics buffer (KB; PBS, 0.1% Tween-20, and 1% bovine serum albumin (BSA)) for 60 s. Then, association to human NKp30 (Abcam) was measured at varying concentrations ranging from 6.25 nM to 200 nM in KB for 300 s, followed by dissociation for 300 s (in KB). In each experiment, one negative control was measured using irrelevant antigen. In addition, one reference value was measured by incubating the antibody in KB instead of the antigen. To analyze the competition with the natural ligand B7-H6 for binding to NKp30, NKp30 was loaded onto anti-his tips (HIS1K) at 3 μg / mL in PBS for 3 min, followed by rinsing the sensors with KB for 60 s. Association of VHH SEED bodies (200 nM) was performed for 300 s (in KB), followed by an additional association step with the natural ligand B7-H6 (500 nM, expressed as a SEED body fusion) for 60 s in KB. Epitope binning experiments of VHH SEED bodies were performed similarly, except that 200 nM was used for both VHH SEED body association steps for 180 s (first association) and 120 s (second association) in KB.

[0569] Data were fitted and analyzed with ForteBio data analysis software 8.0 using a 1:1 binding model after Savitzky-Golay filtering.

[0570] cell culture The EGFR-expressing tumor cell lines A431 and A549 were obtained from DSMZ and cultured in RPMI 1640 Glutamax-I medium or Dulbecco's modified Eagle's medium (R10+ and D10+; all components from Thermo Fisher Scientific) supplemented with 10% FCS, 100 U / ml penicillin, and 100 mg / ml streptomycin, respectively. In addition, Chinese hamster ovary cells (ExpiCHO, Thermo Fisher Scientific) were cultured in suspension in complete ExpiCHO expression medium.

[0571] Tumor cell killing assay Preparations of PBMCs from healthy donors were purchased after receiving written informed consent as previously described (Repp et al., 2011). NK cells were isolated by negative selection using an NK cell isolation kit (Miltenyi Biotech) and cultured at 2x10 in R10+ medium. 6 Cells were maintained at a density of 1000 cells / ml overnight. Cytotoxicity was measured using a standard 4 h immunoassay performed in 96-well microtiter plates in a total volume of 200 μl as described by Repp et al., 2011. 51 Cr release assay. Human PBMCs or purified NK cells were used as effector cells at effector-to-target cell (E:T) ratios of 80:1 and 10:1, respectively. VHH SEED bodies or cetuximab were applied at the indicated concentrations.

[0572] Cytokine release assay Quantification of IFN-γ and TNF-α released by NK cells was performed using the Human Cytokine HTRF Kit (cisbio) as described in Pekar et al., 2021. Briefly, 2.500 viable EGFR-positive A431 cells or EGFR-negative CHO cells per well were seeded in 384 clear-bottom microtiter plates (Greiner Bio-One) and incubated for 3 h. 12.500 viable NK cells were added after overnight incubation in complete medium containing 100 U / ml recombinant human interleukin-2 (R&D systems), resulting in an effector to target cell (E:T) ratio of 5:1. SEED bodies were added to a final concentration of 50 nM. As controls, tumor cells alone as well as NK cells cultured with tumor cells in the absence of NK cell engager were utilized. After 24 h incubation, cells were sedimented by centrifugation and the cytokine-containing supernatant was further processed according to the manufacturer's instructions. The assay plates were measured on a PHERAstar FSX device (BMG Labtech). HTRF optics were used, with excitation at 337 nM and emission at 620 nM and 665 nM. Analysis and fitting of the resulting data was facilitated by MARS software (v.3.32, BMG) following the kit manufacturer's instructions and allowing 4 Parameter Logistic (4PL 1 / y2) model fitting of the standard curve.

[0573] NK cell activation assay For bispecific SEED body-mediated NK cell activation, the early activation marker CD69 was transduced with CD16 高 and CD16 低 CD56 +Cells were detected via flow cytometry. For this purpose, 20.000 tumor cells per well were seeded into 96-well V-bottom microtiter plates (Thermo Fisher Scientific) and incubated for 3 h before the addition of 100.000 NK cells (placed overnight in medium supplemented with 100 U / ml rh IL-2), resulting in an E:T effector to target ratio of 5:1. VHH SEED bodies were added at a final concentration of 50 nM prior to a 24 h incubation period at 37° C. Then, the assay plates were washed twice with PBS+1% BSA and followed by a 1 h incubation step on ice with the detection reagents, namely LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (Thermo Fisher Scientific), anti-human CD56 PE (Miltenyi Biotec), and allophycocyanin-conjugated anti-CD69 (abcam). After repeated washing, cells were analyzed via flow cytometry using the Intellicyt® iQue® Screener Plus (Sartorius) system. For compensation of fluorescent dyes, antibody capture analysis beads (OneComp eBeads™ Compensation Beads, Thermo Fisher Scientific) were employed according to the manufacturer's instructions.

[0574] Data processing and statistical analysis Graphical and statistical analyses were performed with GraphPad Prism 8 software. Repeated measures ANOVA and Bonferroni or Tukey post-hoc tests, as recommended, or Student's t-tests, where appropriate, were employed to calculate P values. p ≤ 0.05 was considered statistically significant. All procedures in Examples 2-7 were carried out as described in Example 1.

[0575] Example 2 Selection of a diverse panel of NKp30-specific VHH single domain antibodies (sdAb) To generate sdAbs targeting NKp30, one llama (Lama glama), one alpaca (Vicugna pacos), and one varizoo (Lama glama x Vicugna pacos) were immunized with recombinant human NKp30 extracellular domain (ECD; produced in-house) (Figure 1A). Yeast surface display libraries were constructed per animal specimen based on PBMCs derived from whole blood, and approximately 5x10 8 Three sub-libraries with sizes ranging from independent clones were obtained respectively. These libraries were subjected to fluorescence-activated cell sorting (FACS)-based selection (Figure 1B). For this purpose, a two-dimensional labeling strategy was exploited to simultaneously detect functional VHH surface expression as well as NKp30 binding. In the first round of selection, each sub-library was sorted individually by FACS using an NKp30 concentration of 1 μM (Figure 1C). The library output was then combined for the second selection round with a significantly reduced NKp30 concentration (100 nM) with the aim of increasing the selection stringency. 96 clones were subsequently sent for sequencing, resulting in 76 unique clones (Figure 1D). From the clonotyping selection output based on CDR3 sequence diversity, 18 clones were chosen for reformatting and expressed as bispecific SEED bodies harboring humanized Fab arms derived from the therapeutic antibody cetuximab targeting EGFR (Wong, 2005; Davis et al., 2010). To suppress Fc-mediated effector functions (eff -), specific amino acid exchanges were introduced into both heavy chains of the bispecific antibody derivative.

[0576] Example 3 NK cell engagers generated based on sdAbs targeting NKp30 demonstrate broad affinity and wide epitope coverage Besides the two SEED bodies that showed no productivity, the expression yields of the bispecific NK cell engagers were at double- to triple-digit milligram scale per liter. Moreover, as shown in Table 1 below, the aggregation properties as indicated by analytical size-exclusion chromatography (SEC) after Protein A purification were highly favorable for most molecules of the target species, i.e., greater than 90%.

[0577] Table 1: Biochemical and biophysical characterization of VHH-based NK cell engagers. [Table 1]

[0578] Interestingly, the bispecific antibodies displayed a wide range of affinities for binding to NKp30, ranging from triple-digit nanomolar binding (VHH6 SEED body) to sub-nanomolar range affinities (VHH1, VHH3, VHH5, VHH8, VHH10, VHH12-VHH16 SEED body). Epitope specificity was determined using biolayer interference (BLI). In the first assay, the competition of VHH SEED bodies with the natural ligand B7-H6 for binding to NKp30 was assessed. For this purpose, recombinant NKp30 ECD was captured on a biosensor. Then, an assembly step was performed employing the respective VHH SEED body, followed by incubation with the N-terminal V-like domain of B7-H6 produced as a SEED body fusion (Pekar et al., 2021). Overall, eight VHH SEED bodies showed competition with B7-H6 for NKp30 binding, indicating that these molecules share the same epitope bins as the natural ligand (Table 1). One partially competing molecule (VHH4 SEED body) as well as seven non-competing moieties were identified. Figure 2A shows representative BLI sensograms for B7-H6 competitors (VHH1 SEED body), non-competitors (VHH2 SEED body), and partial competitors (VHH4 SEED body). Moreover, to more carefully probe the epitope coverage, pairwise competitions were performed using all VHH SEED bodies in all feasible combinations (Table 1, Figure 2B). These experiments revealed that all B7-H6 competing VHH SEED bodies prevented binding to each other and therefore shared very similar epitopes on NKp30. In addition, most of these share overlapping epitopes with SEED bodies that partially compete with B7-H6 for binding to NKp30. However, VHH12 and VHH15 SEED bodies did not compete for binding with VHH4 SEED bodies, also indicating subtle differences in epitope targeting within this series of B7-H6 competitors.Among the bispecifics that do not compete with B7-H6, SEED bodies harboring VHH8, VHH9, VHH10, and VHH11 share the same epitope bins that partially overlap with the bins of VHH2 and VHH6 SEED bodies. Moreover, VHH2, VHH6, and VHH7 SEED bodies were unique in terms of epitope targeting. Taken together, the generated VHH SEED bodies display a wide epitope diversity represented by seven epitope bins.

[0579] Example 4 Killing of EGFR-positive tumor cells by VHH SEED bodies is significantly influenced by the NKp30 binding epitope An initial functional analysis of VHH SEED bodies engaging NK cells was performed in standard chromium release assays using mononuclear cells (MNC) of healthy donors as effector cells and in tumor cell lines A431 and A549 expressing EGFR. Interestingly, B7-H6 competitors and non-competitors were clustered into two groups, with B7-H6 competing VHH SEED bodies mediating more efficient lysis of tumor cells. This was true for the cell line A431 expressing EGFR highly and even more so for the cell line A549 expressing EGFR less (Figure 3). Interestingly, the VHH6 SEED body with the lowest affinity for NKp30 (292 nM) and not interfering with the binding site of the natural ligand B7-H6 elicited the weakest killing of A431 cells in terms of its potency (Figure 3). Moreover, little killing of A549 tumor cells was observed with this molecule. This was in stark contrast to all eight VHH SEED bodies competing with B7-H6 for NKp30 binding. These molecules induced more potent killing than the monoclonal antibody cetuximab, regardless of higher or lower EGFR expression on tumor cells (Figure 3), while activating NK cells exclusively via NKp30. Based on these results, the best three B7-H6 competing SEED bodies (VHH1, VHH5, VHH16 SEED bodies) as well as the best three non-competitive SEED bodies (VHH2, VHH4, VHH8 SEED bodies), encompassing the VHH4 SEED body as a partial competitor, were selected for further characterization. To this end, tumor cell killing assays on A431 and A549 cells using freshly isolated NK cells were performed (Figure 4A; Table 2). Overall, the B7-H6 competitor was more potent than the B7-H6 non-competitor in tumor cell killing, especially using the less EGFR-positive tumor cell line A549, but the dependence of the killing potency of VHH-based NKCE on the targeted epitope on NKp30 was clearly confirmed.

[0580] In a second experiment investigating tumor cell death induced due to natural killer cell activation, the EC50 and maximum killing were determined for an MNC-based killing assay using A431 cells as summarized in Table 2.

[0581] Table 2: [Table 2]

[0582] Example 5 VHH SEED bodies induce potent release of the pro-inflammatory cytokines NF-α and INF-γ In addition, all VHH SEED bodies examined promoted significant NK cell production of TNF-α and IFN-γ in a target-dependent manner (Figure 4B). In this regard, the ability to induce the release of pro-inflammatory cytokines did not significantly differ between non-competitors and competitors of B7-H6. Interestingly, a trend towards the release of elevated levels of TNF-α and IFN-γ was also observed in a direct comparison with the therapeutic antibody cetuximab. While cetuximab induced release of an average of 90.4 pg / ml TNF-α and 336.7 pg / ml IFN-γ (notably, this potency was not statistically significantly higher compared to the monovalent EGFR-targeted effector-inhibited SEED body control (average of 8.6 pg / ml TNF-α and 53.8 pg / ml IFN-γ)), VHH SEED bodies promoted NK cell-mediated release of 103.6-129.2 pg / ml TNF-α and 514.6-752.6 pg / ml IFN-γ, clearly indicating that the cytokine release profiles are distinct. Table 3:

[0583] [Table 3]

[0584] Example 6 Soluble B7-H6 does not impair tumor cell killing induced by VHH SEED bodies Cancer patients unexpectedly have higher soluble B7-H6 (sB7-H6) concentrations in their serum up to 5 nM compared to healthy individuals (Rusakiewicz et al., 2017). To investigate whether shed B7-H6 negatively impacts the killing capacity of generated VHH SEED bodies to engage NK cells, we performed killing assays with B7-H6 competitive VHH1 SEED bodies, partially competitive VHH4 SEED bodies, and non-competitive VHH2 SEED bodies in the presence or absence of a high concentration (178.6 nM) of the extracellular domain of B7-H6 (B7-H6 ECD-His) (Figure 5). Of note, the chosen concentration of 178.6 nM B7-H6 was substantially higher than the concentration typically found in cancer patients in order to probe and even amplify subtle effects that may result from soluble B7-H6-mediated receptor blockade. Interestingly, neither A431 nor A549 cell killing through any of the three VHH SEED bodies was affected by B7-H6 ECD, indicating that B7-H6 given in the serum of cancer patients most likely does not impair the NKCE-mediated tumor cell killing described herein.

[0585] Example 7 Additional FcγRIIIa engagement improves the antitumor activity of VHH SEED bodies In an attempt to further enhance the cytotoxic potential of B7-H6 competing VHH1 and non-competitive VHH2 SEED bodies, we also generated these molecules with Fc regions capable of binding to FcγRIIIa (SEED bodies lacking mutations that abrogate immune effector functions; eff+). As shown in Figure 6, lysis of EGFR-highly expressing A431 tumor cells mediated by B7-H6 competing VHH1 SEED bodies could only be improved by simultaneous engagement of FcγRIIIa in terms of killing efficacy, i.e., maximal lysis, whereas killing potency remained unaffected (EC 50VHH1 SEED body eff- = 2.2pM vs. EC 50 VHH1 SEED body eff+ = 3.2 pM). In contrast, the potency of the B7-H6 non-competitive VHH2 SEED body was significantly increased by 8.8-fold by incorporating an effector-functional Fc region (EC 50 VHH2 SEED body eff- = 34.4pM vs. EC 50 VHH2 SEED body eff+ = 3.9 pM), no significant differences were observed regarding the maximum lysis rates achieved by both VHH2 SEED bodies (Figure 6).

[0586] Taken together, these data suggest that the killing capacity of VHH-based NKCEs may be optimized in some way, i.e., in terms of potency or efficacy, by simultaneous engagement of FcγRIIIa. This beneficial effect contributing to the overall antitumor activity seems to be more pronounced for the B7-H6 non-competitive SEED body, whose tumor cell killing by itself was less potent.

[0587] Example 8 Humanized VHH1 variants Humanized variants of VHH1 (SEQ ID NO: 1) were prepared in which the CDRs are identical to those of VHH1, but compared to individual amino acids of VHH1 in the framework regions, they are replaced by the corresponding amino acids of the human germline sequence (VHH1.1 to VHH1.12, represented by SEQ ID NOs: 67 to 78).

[0588] The constructs were expressed in a small-scale expression as described in Experiment 1. After purification (identity was confirmed by mass verification), the purified variants were analyzed by size-exclusion chromatography (SEC) and their binding to NKp30 and KD values ​​were determined by Biolayer Interferometry (BLI) measurements as described in Experiment 1.

[0589] The results are summarized in the table below: [Table 4]

[0590] These data show that all humanized variants VHH1.1 to VHH1.9 show similar in vitro properties to VHH1 in terms of expression yield / purity, KD, avidity and therefore expected functionality. Among them, VHH1.1 to VHH1.3 match VHH1 particularly well with respect to their in vitro properties. Only VHH1.10 shows an overall less favorable in vitro property (KD and maximum avidity).

[0591] Example 9 Humanized VHH2 variants Humanized variants of VHH2 (SEQ ID NO:2) were prepared in which the CDRs are identical to those of VHH2, but compared to individual amino acids of VHH2 in the framework regions, they are replaced by the corresponding amino acids of the human germline sequence (VHH2.1 to VHH2.18, represented by SEQ ID NOs:79 to 96).

[0592] The constructs were expressed in a small-scale expression as described in Experiment 1. After purification (identity was confirmed by mass verification), the purified variants were analyzed by size-exclusion chromatography (SEC) and their binding to NKp30 and KD values ​​were determined by Biolayer Interferometry (BLI) measurements as described in Experiment 1.

[0593] The results are summarized in the table below: [Table 5]

[0594] From these data, it can be seen that all of the humanized variants VHH2.1, VHH2.2, VHH2.3, VHH2.5, VHH2.6, VHH2.7, VHH2.8, VHH2.11, VHH2.12, VHH2.15, and VHH2.16 show similar in vitro properties to VHH1 in terms of expression yield / purity, KD, avidity, and therefore expected functionality. Among them, VHH2.1, VHH2.2, VHH2.5, VHH2.8, VHH2.11, VHH2.12, and VHH2.16 match VHH2 particularly well with respect to their in vitro properties. VHH2.4, VHH2.9, VHH2.10, VHH2.13, VHH2.14, VHH2.17 and VHH2.18 show overall less favorable in vitro properties (KD and maximum avidity).

[0595] References [Table 6-1]

[0596] [Table 6-2]

[0597] [Table 6-3]

[0598] [Table 6-4]

Claims

1. A compound comprising a VHH antibody domain or a fragment thereof, wherein (a) the VHH antibody domain or a fragment thereof comprises one of the complementarity determining regions CDR1, CDR2, and CDR3 of VHH1 to VHH16 as shown in the following CDR table; (b) the VHH antibody domain or a fragment thereof comprises the complementarity determining regions CDR1, CDR2, and CDR3 as defined in (a) with modifications, wherein the modification is that at least one sequence of CDR1, CDR2, and CDR3 is humanized; or (c) the VHH antibody domain or a fragment thereof comprises the complementarity determining regions CDR1, CDR2, and CDR3 as defined in (a) with modifications, wherein the modification is - replacement, addition, or deletion up to a maximum of 3 amino acids in CDR1, - replacement, addition, or deletion up to a maximum of 3 amino acids in CDR2, and / or - replacement, addition, or deletion up to a maximum of 3 amino acids in CDR3; CDR table: 【Table 1-1】 【Table 1-2】

2. The compound comprises a sequence represented by SEQ ID NO: 1 (or a fragment thereof) having one or more amino acid substitutions as compared to the sequence of SEQ ID NO: 1, wherein, when such amino acid substitution(s) is / are located outside the CDRs, the amino acid substitution(s) is / are selected from the following: D1E, Q5V, L11V, T24A, E44G, H45L, G47W, F70I, V79L, K87R, P88A, Q116L. The compound according to Claim 1.

3. The compound according to Claim 1, having a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 77, or a fragment thereof.

4. The compound comprises a sequence represented by SEQ ID NO: 2 (or a fragment thereof) having one or more amino acid substitutions as compared to the sequence of SEQ ID NO: 2, Here, when such amino acid substitution(s) is / are positioned outside the CDR, the amino acid substitution(s) is / are selected from the following: D1E, Q5V, L11V, A14P, V24A, F37V, A44G, R45L, S47W, V79L, K87R, P88A, Q118L, and the compound according to claim 1.

5. The compound according to claim 1, wherein the compound comprises a sequence represented by any one of the following SEQ ID NOs: SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 93, SEQ ID NO: 94, or a fragment thereof.

6. A compound comprising a VHH antibody domain or a fragment thereof, wherein (A) the VHH antibody domain comprises any one of VHH1 to VHH16 shown in the table of VHH sequences below; (B) the VHH antibody domain comprises a VHH sequence as defined in (A) with modifications, wherein the modification is that the sequence is humanized; (C) the VHH antibody domain comprises a VHH sequence as defined in (A) with modifications, wherein the modification is a replacement, addition, or deletion of up to 25 amino acids; or (D) the VHH antibody domain comprises a VHH sequence that is at least 75% identical to the VHH sequence mentioned in (A). Table of VHH sequences: 【Table 2-1】 【Table 2-2】 【Table 2-3】

7. The VHH antibody domain comprises VHH1 shown in the table of VHH sequences or a fragment thereof with additional modifications of (B) or (C), wherein the modification of (B) or (C) consists of substitutions of one or more amino acids as compared to the sequence of SEQ ID NO: 1, and wherein the amino acid substitution(s) is / are selected from the following: D1E, Q5V, L11V, T24A, E44G, H45L, G47W, F70I, V79L, K87R, P88A, Q116L, and the compound according to claim 6.

8. The VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 77, or a fragment thereof, and the compound according to claim 6.

9. The VHH antibody domain comprises VHH1 or a fragment thereof shown in the table of VHH sequences with additional modifications of (B) or (C), wherein the modification of (B) or (C) consists of one or more amino acid substitutions as compared to the sequence of SEQ ID NO: 2, wherein the amino acid substitution(s) is / are selected from the following: D1E, Q5V, L11V, A14P, V24A, F37V, A44G, R45L, S47W, V79L, K87R, P88A, Q118L; the compound according to claim 6.

10. The VHH antibody domain comprises a VHH sequence represented by any one of the following SEQ ID NOs: SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 93, SEQ ID NO: 94, or a fragment thereof; the compound according to claim 6.

11. The compound binds to NKp30 with an affinity of at least about 1×10 6 M -1 in a binding assay by BLI (Biolayer Interferometry) with the recombinant extracellular domain of NKp30 (NKp30-ECD, SEQ ID NO: 66) in KB buffer (PBS + 0.1% Tween-20 + 1% BSA), the compound according to any one of claims 1 to 10.

12. The compound further comprises a targeting component capable of specifically binding to a tumor-associated antigen; the compound according to any one of claims 1 to 10.

13. A pharmaceutical composition comprising the compound according to any one of claims 1 to 10.

14. A medicament comprising the pharmaceutical composition according to claim 13.

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