WO2025168114A1

WO2025168114A1 - Multifunctional nk cell engager

Info

Publication number: WO2025168114A1
Application number: PCT/CN2025/076494
Authority: WO
Inventors: Mengting Chen; Yinhui DING; Shiyong GONG; Huifeng LV; Zelong MA; Lijun Wang; Danqing WU; Xuan WU; Rui Zhang
Original assignee: Shanghai Epimab Biotherapeutics Co Ltd
Current assignee: Shanghai Epimab Biotherapeutics Co Ltd
Priority date: 2024-02-09
Filing date: 2025-02-08
Publication date: 2025-08-14
Anticipated expiration: 2026-08-09

Abstract

Provided are proteins capable of engaging NK cells in cancer therapy, especially multi-specific proteins capable of binding IL 15 receptor, NKp46, target antigen of interest, and optionally Fc gamma receptor such as CD16a. Also provided are NKp46-binding proteins. The proteins have utility in the treatment of diseases, such as cancer.

Description

Multifunctional NK cell engager

Cross-reference to related applications

This application is based on the PCT application with Application No. PCT/CN2024/077127, filed on February 9, 2024, and PCT/CN2024/130521 filed on November 7, 2024, and claims priority to the aforementioned applications, the entire contents of which are hereby incorporated by reference into this application.

Field of the Invention

The present disclosure relates to proteins capable of engaging NK cells in cancer therapy, especially multi-specific proteins capable of binding IL15 receptor, NKp46, target antigen of interest, and optionally Fc gamma receptor such as CD16a. The present disclosure also relates to NKp46-binding proteins. The proteins according to the present disclosure have utility in the treatment of diseases such as cancer.

Background

Interleukin 15 (IL15 or IL-15) is one example of a pluripotent cytokine that acts on a cytokine receptor expressed by NK cells. IL-15 binds to the IL-15 receptor (IL-15R) which is composed of three subunits: IL-15Ra, CD122, and CD132. IL-15Ra (CD215) specifically binds IL15 with very high affinity and is capable of binding IL-15 independently of other subunits, while CD122 and CD132 are shared with the receptor for IL-2.

Natural killer (NK) cells are a type of lymphocytes and belong to innate immune system, accounting for 5-20%of peripheral blood lymphocytes in human. Human NK cell activation and function are regulated by a balance between inhibitory receptors and activating receptors recognizing stressed cells, virus-infected cells, opsonized cells, or tumor cells, allowing for a much faster immune reaction.

NK cells play an important role in cancer immunosurveillance in all stages of cancer development. Cancer cells can escape T-cell responses by losing major histocompatibility complex (MHC) /human leucocyte antigen (HLA) class I molecules, which is a major kind of inhibitory receptor ligands of NK cells. Afterwards, NK cells can discriminate MHC negative 'missing-self' cancer cells and eliminate those cells by the mechanism of breaking the balance towards activation receptors and leading to NK cell activation and tumor killing.

As NK cells have undisputable roles in controlling cancer development and metastasis, to date, several multiple NK activation receptor binding proteins were reported, utilizing NK activation receptors CD16a, NKG2D, or NKp46 to activate and direct NK cells to tumor cells for cancer therapy.

NKp46 is the major triggering receptor involved in the natural cytotoxicity of human NK cells which can induce strong cell triggering leading to [Ca²⁺] increases, cytokine production, and cytolytic activity. NK cells in the tumor microenvironment down-regulate multiple activation receptors such as CD16a, NKG2D, and NKp46, where NKp46 downregulation is limited, while NKG2D and CD16a is more dramatical.

Some studies have found multi-specific proteins that specifically bind IL-2 and NKp46, and thus lyse a target cell of interest via multiple receptors (WO2022258662) . However, there remains a need in developing multi-specific proteins to engage NK cells in cancer therapy, particularly those that can provide therapeutic advantages over conventional monoclonal antibodies.

Summary of Invention

The present disclosure provides a multi-specific protein that specifically binds to IL-15 receptor, NKp46, a target antigen, and optionally to Fc gamma receptor, such as Fcγ RIII, e.g., CD16a. In some embodiments, said target antigen is a tumor/cancer antigen, such as tumor-associated antigen (TAA) , or tumor specific antigen (TSA) .

In some embodiments, the multi-specific protein comprises three antigen binding portions (ABPs) , one specifically binds to IL-15 receptor, and other two are antigen binding portions (ABPs) specifically binds to antigens A and B, respectively; wherein one of A and B is NKp46 while the other is a target antigen different from IL-15 receptor and NKp46.

The multi-specific protein provided herein have improved properties, such as high NK cell selectivity and high stability. It is also easy for preparation and/or purification and has high yield and/or purity.

The present disclosure also provides a new NKp46 binding protein, especially an antibody or antigen binding fragment that specifically binds NKp46.

The present disclosure also provides nucleic acids encoding the multi-specific protein or the NKp46 binding protein, the vector and host cell comprising said nucleic acids, and a method to prepare the multi-specific protein or the NKp46 binding protein.

The present disclosure further provides method and use of the the multi-specific protein or the NKp46 binding protein, e.g., in the treatment of diseases, such as cancer.

Brief Description of the Drawings

Figure 1: NK cell activation by chimeric (A) and humanized (B) anti-NKp46 antibodies.

Figure 2: Exemplary domain configuration of IL15 variants (IL15v) .

Figure 3: Exemplary dose-response curves for IL15v potency determination.

Figure 4: Domain configurations of Format I.

Figure 5: Domain configurations of Format II.

Figure 6: Domain configurations of Format III.

Figure 7: Multi-specific proteins in Format III-1 with various linkers.

Figure 8: Domain configurations of Format IV.

Figure 9: Domain configurations of Format T5.

Figure 10: IL15 potency of multi-specific proteins in different formats.

Figure 11: Representative dose-response curves for IL15 activities of selected multi-specific proteins.

Figure 12: IL15 potency for multi-specific proteins targeting different cancer antigens.

Figure 13: Marginal IL15 activity for CD8+ and CD4+ T cells.

Figure 14: NK cytotoxicities of selected multi-specific proteins.

Figure 15: In vivo anti-tumor efficacy of EM1030-NIH-N11 for B16F10-Her2 engrafted model in B6-hNKp46 knock in mice.

Figure 16: Cytokine release of EM1030-NIH-N11 in comparison with IL15-N5.

Figure 17: Cytokine release of EM1030-NIC123-N1 in comparison with talacotuzumab, MGD024, and IPH6101 analogs.

Detailed Description

Definitions

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of "or" means "and/or" unless stated otherwise. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

The term “about” used in conjunction with a numerical value is intended to encompass the numerical values in a range from a lower limit less than the specified numerical value by 5%to an upper limit greater than the specified numerical value by 5%.

As used herein, the term “and/or” refers to any one of the options or any two or more of the options.

As used herein, the term “comprise” , “include” or the variants thereof is intended to mean that the described elements, integers or steps are included, but not to the exclusion of any other elements, integers or steps. The term “comprise” or “include” used herein, unless otherwise specified, also encompasses the situation where the entirety consists of the described elements, integers or steps. For example, when referring to an antibody variable region “comprising” a particular sequence, it is also intended to encompass an antibody variable region consisting of the particular sequence.

The extracellular domain of IL-15Rα comprises a domain referred to as the “sushi domain” , which binds IL-15. The general sushi domain, also referred to as complement control protein (CCP) modules or short consensus repeats (SCR) , is a protein domain found in several proteins, including multiple members of the complement system. The sushi domain adopts a beta-sandwich fold, which is bounded by the first and fourth cysteine of four highly conserved cysteine residues, comprising a sequence stretch of approximately 60 amino acids (Norman, Barlow, et al. J Mol Biol. 1991; 219 (4) : 717-25) . The amino acid residues bounded by the first and fourth cysteines of the sushi domain IL-15Ra comprise a 62 amino acid polypeptide referred to as the minimal domain. Including additional amino acids of IL-15Rα at the N-and C-terminus of the minimal sushi domain, such as inclusion of N-terminal lie and Thr and C-terminal lie and Arg residues result in a 65 amino acid extended sushi domain.

The term “IL15 moiety” as mentioned herein comprises an IL15, with optionally a IL15Rα or its sushi domain. The term “IL15/IL15Rα complex” , as used herein, refers to a recombinant protein comprising an IL-15 domain non-covalently complexed with an IL15 receptor alpha (IL15Rα) or its sushi domain, and optionally stabilized by an interchain-di-sulfide bond therebetween. In some embodiments, said complex may be fused, with or without a linker, to N-terminus or C-terminus of an immunoglobulin Fc domain, constituting the IL15 moiety of the multi-specific proteins described herein. In some embodiments, if the IL15Rα sushi domain is complexed with IL15, the “IL15/IL15Rαcomplex” is also named as “IL-15 sushi complex” or “IL15/IL15RαSu complex” .

The term “NKp46” used herein refers to a protein or polypeptide encoded by the Ncr1 gene or by a cDNA prepared from such a gene. NKp46 is the major triggering receptor involved in the natural cytotoxicity of human NK cells which can induce strong cell triggering leading to [Ca²⁺] increases, cytokine production, and cytolytic activity. NK cells in the tumor microenvironment down-regulate multiple activation receptors such as CD16a, NKG2D, and NKp46, where NKp46 downregulation is limited, while NKG2D and CD16a is more dramatical. The term used herein includes the wildtype NKp46 and the variant or alleles thereof. In one embodiment, illustrated amino acid sequence of human NKp46 (isoform4) corresponds to NCBI accession number O76036.1. NKp46 also covers the functional domain of the protein, e.g., the Extra Cellular Domain ECD in some embodiments is also covered by the term.

The term “anti-NKp46 antibody” , “anti-NKp46” , “NKp46 antibody” or “antibody that binds to NKp46” used herein refers to an antibody capable of binding to (human) NKp46 with sufficient affinity such that the antibody can be used as a therapeutic agent targeting (human) NKp46. In one embodiment, the (human) NKp46 antibody binds to (human) NKp46 with high affinity in vitro or in vivo. In one embodiment, the (human) NKp46 antibody binds to cells expressing NKp46. In some embodiments, the binding is determined, e.g., by radioimmunoassay (RIA) , bio-layer interferometry (BLI) , MSD assay, surface plasmon resonance (SPR) or flow cytometry.

The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state, is substantially free of other proteins from the same species, is expressed by a cell from a different species, or does not occur in nature. A polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates may be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

The term “specific binding” or “specifically binding” in reference to the interaction of an antibody, a binding protein, or a peptide with a second chemical species, means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the second chemical species. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. In general, if an antibody is specific for epitope “A” , the presence of a molecule containing epitope A (or free, unlabeled A) , in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

The term “antibody” broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art and non-limiting embodiments are discussed below.

In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains: CH1, CH2, and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) , interspersed with regions that are more conserved, termed framework regions (FRs) . Each VH and VL is comprised of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. First, second and third CDRs of a VH domain are commonly enumerated as CDR-H1, CDR-H2, and CDR-H3; likewise, first, second and third CDRs of a VL domain are commonly enumerated as CDR-L1, CDR-L2, and CDR-L3. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

The term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, i.e., a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain, for example, as in the case of the Fc regions of IgM and IgE antibodies. The Fc region of IgG, IgA, and IgD antibodies comprises a hinge region, a CH2 domain, and a CH3 domain. In contrast, the Fc region of IgM and IgE antibodies lacks a hinge region but comprises a CH2 domain, a CH3 domain and a CH4 domain. Variant Fc regions having substitutions of amino acid residues in the Fc portion to alter antibody effector function are known in the art (see, e.g., Winter et al., US Patent Nos. 5, 648, 260 and 5, 624, 821) . The Fc portion of an antibody may mediate one or more effector functions, for example, cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) , and/or half-life/clearance rate of antibody and antigen-antibody complexes. In some cases, these effector functions are desirable for therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRs and complement C1q, respectively. In still another embodiment at least one amino acid residue is replaced in the constant region of the antibody, for example the Fc region of the antibody, such that effector functions of the antibody are altered. The dimerization of two identical heavy chains of an immunoglobulin is mediated by the dimerization of CH3 domains and is stabilized by the disulfide bonds within the hinge region that connects CH1 constant domains to the Fc constant domains (e.g., CH2 and CH3) . The anti-inflammatory activity of IgG is dependent on sialylation of the N-linked glycan of the IgG Fc fragment. The precise glycan requirements for anti-inflammatory activity have been determined, such that an appropriate IgG1 Fc fragment can be created, thereby generating a fully recombinant, sialylated IgG1 Fc with greatly enhanced potency (see, Anthony et al., Science, 320: 373-376 (2008) ) .

The terms “antigen-binding portion (ABP) ” , “antigen-binding domain (ABD) ” and “antigen-binding fragment” or “functional fragment” of an antibody are used interchangeably and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, i.e., the same antigen as the full-length antibody from which the portion or fragment is derived. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens (e.g., NKp46 and a different antigen, such as HER2, LIV-1, CD228, CD20, ALPP/ALPPL2, CD123, or TROP2) . Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F (ab’) ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature, 341: 544-546 (1989) ; PCT Publication No. WO 90/05144) , which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR) . Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) ; see, for example, Bird et al., Science, 242: 423-426 (1988) ; and Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988) ) . Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody and equivalent terms given above. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) . Such antibody binding portions are known in the art (Kontermann and Dübel eds., Antibody Engineering (Springer-Verlag, New York, 2001) , p. 790 (ISBN 3-540-41354-5) ) . In addition, single chain antibodies also include “linear antibodies” comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding portions (Zapata et al., Protein Eng., 8 (10) : 1057-1062 (1995) ; and US Patent No. 5,641,870) ) .

An immunoglobulin constant I domain refers to a heavy (CH) or light (CL) chain constant domain. Murine and human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.

The term “monoclonal antibody” or “mAb” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method.

The term “VHH” , “nanobody” or “sdAb” as used herein is interchangable, referring to a single heavy chain variable domain antibody devoid of light chains. Typically, a VHH is an antibody of the type that may be present in Camelidae or cartilaginous fish that are naturally devoid of light chains or is a synthetic and non-immunized VHH that can be artificially constructed. Each heavy chain comprises a variable region encoded by V-, D-and J exons. Said VHH may be a natural VHH antibody, typically a Camelid antibody, or a recombinant protein comprising a heavy chain variable domain.

The term “linker” as used herein refers to any molecule that enables direct attachment of different parts of a binding protein, especially a multi-specific protein. Examples of linkers to establish covalent linkages between different parts of the multi-specific protein include peptide linkers and non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG) , polypropylene glycol, polyalkylene oxide, or copolymers of polyethylene glycol, polypropylene glycol. In some embodiments, the term “peptide linker” according to the invention refers to a sequence of amino acids, wherein said sequence links together the amino acid sequences of the various parts of the multi-specific antibody. Preferably, the peptide linker has a length sufficient to link the two entities in such a way that they maintain their conformation relative to each other so as not to interfere with the desired activity. The peptide linker may or may not comprise predominantly the following amino acid residues: Gly, Ser, Ala or Thr. Useful linkers include glycine-serine polymers including, for example, (GS) n, (GSGGS) n, (GGGGS) n, (GGGS) n, (GGGGS) nG, (GSSS) n, (GSSS) nGS and the likes, where n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10) . Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.

The term “human sequence” , in relation to the light chain constant domain CL, heavy chain constant domain CH, and Fc region of the antibody or the binding protein according to the present disclosure, means the sequence is of, or from, human immunoglobulin sequence. The human sequence of the present disclosure may be native human sequence, or a variant thereof including one or more (for example, up to 20, 15, 10) amino acid residue changes.

The term “chimeric antibody” refers to antibodies that comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies that comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having human heavy and light chain variable regions in which one or more of the human CDRs has been replaced with murine CDR sequences.

The term “humanized antibody” refers to antibodies that comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like” , i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which CDR sequences from a non-human species (e.g., mouse) are introduced into human VH and VL framework sequences. A humanized antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises framework regions and constant regions having substantially the amino acid sequence of a human antibody but complementarity determining regions (CDRs) having substantially the amino acid sequence of a non-human antibody. As used herein, the term “substantially” in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%or at least 99%identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially at least one, and typically two, variable domains (Fab, Fab’, F (ab’) ₂, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In an embodiment, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and/or humanized heavy chain.

A humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including without limitation IgG1, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well known in the art.

The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the acceptor framework may be mutagenized by substitution, insertion and/or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. In an exemplary embodiment, such mutations, however, will not be extensive. Usually, at least 80%, at least 85%, at least 90%, or at least 95%of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. Back mutation at a particular framework position to restore the same amino acid that appears at that position in the donor antibody is often utilized to preserve a particular loop structure or to correctly orient the CDR sequences for contact with target antigen.

The term “target antigen” refers to an antigen of interest that the multi-specific protein or antibody of the present disclosure targets to bind. Typically, the target antigen is expressed by detrimental cells that may cause diseases or disorders; in some embodiments, the target antigen may be, for example, a tumor/cancer antigen, a viral antigen, a bacterial antigen, etc. In certain embodiments, said target antigen is a tumor/cancer antigen, for example, a tumor-associated antigen (TAA) , or tumor-specific antigen (TSA) .

The term “tumor-associated antigen" or “TAA” is meant to be one or more molecules (typically protein, carbohydrate, lipid or combination thereof) expressed on the surface of a tumor/cancer cell, either entirely or as a fragment (e.g., peptide-MHC) , and useful for the preferential targeting of a medicament to the tumor/cancer cell. In some embodiments, a TAA is a marker expressed by both normal cells and cancer cells; in some embodiments, a TAA is a cell surface molecule overexpressed in a tumor/cancer cell in comparison to a normal cell. In some embodiments, a TAA is a cell surface molecule that is inappropriately synthesized in the tumor/cancer cell, for instance, a molecule containing deletions, additions or mutations in comparison to those expressed on a normal cell. In some embodiments, a TAA will be expressed exclusively on the cell surface of a tumor/cancer cell, entirely or as a fragment, and not synthesized or expressed by a normal cell. Thereby, the term “TAA” encompasses antigens that are specific to tumor/cancer cells, sometimes also annoted as tumor-specific antigens ( “TSAs” ) .

An “antibody that binds to the same or overlapping epitope” as a reference antibody refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%or 95%or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90%or 95%or more of the binding of the antibody to its antigen in a competition assay.

An antibody that competes with a reference antibody for binding to its antigen refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%or 95%or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90%or 95%or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether an antibody competes with another antibody, such as direct or indirect solid-phase radioimmunoassay (RIA) , direct or indirect solid-phase enzyme immunoassay (EIA) and sandwich competition assay.

An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits 50%, 60%, 70%, 80%, 90%or 95%or more of the binding of the reference antibody to its antigen. Conversely, the reference antibody inhibits 50%, 60%, 70%, 80%, 90%or 95%or more of the binding of the antibody to its antigen. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant) . Methods for determining affinity are known in the art.

An antibody that shows the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody that is capable of having at least 50%, 60%, 70%, 80%, 90%or 95%or more of the binding affinity and/or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and/or specificity.

“Complementarity determining region” or “CDR region” or “CDR” is a region in an antibody variable domain that is highly variable in sequence and forms a structurally defined loop ( “hypervariable loop” ) and/or comprises antigen-contacting residues ( “antigen contact site” ) . CDRs are primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are generally referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the heavy chain variable domain of the antibody are referred to as CDR-H1, CDR-H2 and CDR-H3, whereas the CDRs located in the light chain variable domain of the antibody are referred to as CDR-L1, CDR-L2 and CDR-L3. In a given amino acid sequence of a light chain variable region or a heavy chain variable region, the exact amino acid sequence boundary of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems including, e.g., Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature, 342: 877-883; Al-Lazikani et al., Standard conformations for the canonical structures of immunoglobulins, Journal of Molecular Biology, 273: 927-948 (1997) ) , Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4^th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987) ) , AbM (University of Bath) , Contact (University College London) , International ImMunoGeneTics database (IMGT) (imgt. cines. fr/on the World Wide Web) , and North CDR definition based on the affinity propagation clustering using a large number of crystal structures.

For example, according to different CDR determination schemes, the residues of each CDR are as follows:

Unless otherwise indicated, in the present disclosure, the term “CDR” or “CDR sequence” encompasses CDR sequences determined in any of the ways described above. CDRs may also be determined based on the same Kabat numbering position as a reference CDR sequence (e.g., any one of the exemplary CDRs of the invention) .

As used herein, the amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5^th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and is referred to as “numbering according to Kabat” herein. Specifically, the Kabat numbering system (see pages 647-660) of Kabat, et al., Sequences of Proteins of Immunological Interest, 5^th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) is used for the light chain constant domain CL of kappa and lambda isotype, and the Kabat EU index numbering system (see pages 661-723) is used for the constant heavy chain domains (CH1, Hinge, CH2 and CH3, which is herein further clarified by referring to “numbering according to Kabat EU index” in this case) .

General information regarding the sequences of human immunoglobulins light and heavy chains is given in: Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5^th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) .

In one embodiment, the variable region CDRs of the antibody of the present disclosure are determined based on Kabat Scheme.

It should be noted that boundaries of CDRs in variable regions of an antibody determined by different assignment systems may differ. That is, the CDR sequences of variable regions of the same antibody defined by different assignment systems differ. Accordingly, when it comes to defining an antibody with specific CDR sequences defined in the present disclosure, the scope of antibody also encompasses such antibodies whose variable region sequences comprise the specific CDR sequences, but have claimed CDR boundaries different from the specific CDR boundaries defined by the present disclosure due to a different scheme (e.g., different assignment system rules or their combinations) applied.

In one embodiment, the human IgG heavy chain Fc region is from Asp221 or from Cys226 or from Asp231 to the C-terminus of heavy chain.

The term “multi-specific protein” or “multi-specific binding protein” can be used interchangeably referring to a binding protein capable of binding two or more targets of different specificity. Multi-specific protein can be bispecific, trispecific or tetraspecific binding proteins.

“Immunoconjugate” is an antibody conjugated to one or more other substances, including but not limited to active ingredient.

The term “therapeutic agent” used herein encompasses any substance that is effective in preventing or treating a tumor, e.g., cancer, including a chemotherapeutic agent, a cytokine, a cytotoxic agent, an antibody of different specificity or functional fragment thereof, a small molecule drug or an immunosuppressant (e.g., an immunosuppressive agent) .

The term “cytotoxic agent” used herein refers to a substance that inhibits or prevents cell functions and/or causes cell death or cell destruction.

“Chemotherapeutic agents” include chemical compounds useful in the treatment of immune system diseases.

The term “small molecule drug” refers to a low molecular weight organic compound capable of regulating biological processes. “Small molecule” is defined as a molecule with a molecular weight of less than 10 Kd, usually less than 2 Kd and preferably less than 1 Kd.The small molecule includes but is not limited to inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules penetrate cells better, are less susceptible to degradation and are less likely to induce an immune response compared with large molecules.

The term “immunomodulatory agent” used herein refers to a natural or synthetic active agent or drug that suppresses or modulates an immune response. The immune response may be a humoral response or a cellular response. The immunomodulatory agent includes an immunosuppressant.

“Immunosuppressant” , “immunosuppressive drug” or “immunosuppressive substance” used herein refers to a therapeutic agent used in immunosuppressive therapy to suppress or prevent the activity of the immune system.

As used herein, the term “effective amount” refers to the amount of a therapy that is sufficient to reduce or ameliorate the severity and/or duration of a disorder or one or more symptoms thereof; prevent the advancement of a disorder; cause regression of a disorder; prevent the recurrence, development, or progression of one or more symptoms associated with a disorder; detect a disorder; or enhance or improve the prophylactic or therapeutic effect (s) of another therapy (e.g., prophylactic or therapeutic agent) .

The term “recombinant host cell” (or simply “host cell” ) , is intended to refer to a cell into which exogenous DNA has been introduced. In an embodiment, the host cell comprises two or more (e.g., multiple) nucleic acids encoding antibodies, such as the host cells described in US Patent No. 7,262,028, for example. Such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. In an embodiment, host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life. In another embodiment, eukaryotic cells include protist, fungal, plant and animal cells. In another embodiment, host cells include but are not limited to the prokaryotic cell line Escherichia coli; mammalian cell lines CHO, HEK 293, COS, NS0, SP2 and PER. C6; the insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae.

The term “label” used herein refers to a compound or composition which is directly or indirectly conjugated or fused to an agent, such as a polynucleotide probe or an antibody, and facilitates the detection of the agent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change to a detectable substrate compound or composition in the case of enzymatic labeling. The term is intended to encompass direct labeling of a probe or an antibody by coupling (i.e., physical linking) a detectable substance to the probe or an antibody and indirect labeling of a probe or antibody by reacting with another reagent which is directly labeled.

“Individual” or “subject” includes mammals. The mammals include, but are not limited to, domestic animals (e.g., cattle, goats, cats, dogs, and horses) , primates (e.g., human and non-human primates such as monkeys) , rabbits, and rodents (e.g., mice and rats) . In some embodiments, the individual or subject is a human.

An “isolated” antibody is an antibody which has been separated from components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95%or 99%as determined by, e.g., electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF) and capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC) .

“An isolated nucleic acid encoding an anti-NKp46 antibody or a fragment thereof” refers to one or more nucleic acid molecules encoding the antibody heavy or light chain (or fragment thereof, e.g., heavy chain variable region or light chain variable region) , including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

“Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm needed to achieve maximal alignment over the full length of the sequences being compared.

When percentages of sequence identity are referred to in this application, these percentages are calculated relative to the full length of the longer sequence, unless otherwise specifically indicated. The calculation relative to the full length of the longer sequence applies to both the nucleic acid sequence and the polypeptide sequence. The term “anti-tumor effect” refers to a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, decrease in tumor volume, decrease in number of tumor cells, decrease in tumor cell proliferation, or decrease in tumor cell viability.

The terms “cancer” and “cancerous” refer to or describe a physiological disease in mammals that is typically characterized by unregulated cell growth. In certain embodiments, cancers suitable for treatment with the antibody of the present disclosure include metastatic forms of such cancers. The cancer can be solid cancer or hematopoietic cancer, e.g., a cancer of a tissue selected from the group consisting of colon, rectum, cervix, oropharynx, nasopharynx, liver, stomach, head and neck, oral cavity, oesophagus, lip, mouth, tongue, tonsil, nose, throat, salivary gland, sinus, pharynx, larynx, prostate, lung, bladder, skin, kidney, ovary or mesothelium.

The term “tumor” refers to all neoplastic cell growth and proliferation, whether being malignant or benign, and all pre-cancerous and cancerous cells and tissues. Tumor includes both solid or hematopoietic tumors.

The terms “cancer” , “cancerous” and “tumor” are not mutually exclusive when referred to herein.

The term “pharmaceutical acceptable supplementary material” refers to diluents, adjuvants (e.g., Freund’s adjuvants (complete and incomplete) ) , excipients, carriers, stabilizers, or the like, which are administered with the active substance.

The term “pharmaceutical composition” refers to such a composition that exists in a form allowing effective biological activity of the active ingredient contained therein, and does not contain additional ingredients having unacceptable toxicity to a subject to which the composition is administered.

The term “combination product” or “pharmaceutical combination” refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit and a pharmaceutical composition. The term “non-fixed combination” means that the active ingredients (e.g., (i) an anti-NKp46 antibody or a fragment thereof, and (ii) an additional therapeutic agent) are administered, either simultaneously or sequentially (without specific time limitation or at identical or different time intervals) , to a patient as separate entities, wherein such administration provides two or more prophylactically or therapeutically effective active agents in the patient. In some embodiments, the anti-NKp46 antibody or the fragment thereof and the additional therapeutic agent used in the pharmaceutical combination are administered at levels that do not exceed their levels when used alone. The term “fixed combination” means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dose and/or time intervals of two or more active agents are preferably selected such that the combined use of the components can result in a therapeutic effect on the disease or disorder which is greater than that achieved by the use of either component alone. The ingredients may each take a separate formulation form and such separate formulation forms may be the same or different.

The term “combination therapy” refers to the administration of two or more therapeutic agents or modalities (e.g., radiotherapy or surgery) to treat the diseases as described herein. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, for example, in a single capsule with a fixed proportion of active ingredients. Alternatively, such administration includes co-administration of the active ingredients in a variety of or separate containers (such as tablets, capsules, powder and liquid) . The powder and/or liquid can be reconstituted or diluted to a desired dose before administration. In addition, such administration also includes using each type of the therapeutic agents at approximately the same time or in a sequential manner at different times. In any case, the therapeutic regimen will provide the beneficial effect of the pharmaceutical combination in the treatment of disorders or symptoms described herein.

As used herein, “treatment” (or “treat” or “treating” ) refers to slowing, interrupting, arresting, alleviating, stopping, lowering, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

As used herein, “prevention” (or “prevent” or “preventing” ) includes the inhibition of the development or progression of symptoms of a disease or disorder, or a specific disease or disorder. In some embodiments, subjects with family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of a cancer, particularly in subjects at risk of cancer.

As used herein, the term “administering” , “administration” or “administered” refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.

The term “sample” from subject/patient is meant a collection of cells or fluids obtained from a cancer patient or cancer subject. The source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject. The tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like. Examples of tumor samples herein include, but are not limited to, tumor biopsy, fine needle aspirate, bronchiolar lavage, pleural fluid, sputum, urine, a surgical specimen, circulating tumor cells, serum, plasma, circulating plasma proteins, ascitic fluid, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, as well as preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.

The term “vector” , as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid” , which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) . Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors” ) . In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the present disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) , which serve equivalent functions.

“Subject/patient/individual sample” as used herein, refers to a collection of cells or fluids obtained from a patient or a subject. The source of tissue or cell samples can be solid tissues, e.g., from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids such as cerebrospinal fluids, amniotic fluids, peritoneal fluids, or interstitial fluids; and cells from a subject at any time during pregnancy or development. Tissue samples may comprise compounds which are naturally not mixed with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients and antibiotics.

Multi-specific proteins

The present disclosure provides a multi-specific protein that specifically binds to IL-15 receptor, NKp46, a target antigen and optionally to Fc gamma receptor, such as Fcγ RIII, e.g., CD16a. In some embodiments, said target antigen is a tumor/cancer antigen, such as tumor-associated antigen (TAA) , or tumor specific antigen (TSA) .

In certain embodiments, the binding portion that specially binds to IL-15 receptor comprises or consists of an IL-15 protein or its functional fragment, or an IL15-IL15Rαcomplex or IL15-sushi complex.

In certain embodiments, the ABP that specifically binds to NKp46 is derived from an NKp46 antibody, e.g., a Fab fragment, Fv fragment, scFv fragment, or a VHH of an NKp46 antibody.

In certain embodiments, the ABP that specifically binds to a target antigen is derived from an antibody or its binding fragment having specificity binding to the target antigen, e.g., a Fab , Fv, scFv, or VHH of said target-antigen antibody.

In some embodiments, the multi-specific protein of the present disclosure further comprises two Fc chains.

In some embodiments, the configuration of the multi-specific proteins is illustrated in Figures 4-9.

In some embodiments, the multi-specific proteins in the present disclosure are configured in Formats I-IV and T5.

In Format I, as exemplified by I-1, I-2, and I-3 (Figure 4) , IL-15 is placed on N-terminus of Fc domain containing Fc-engineered mutations favoring heterodimerization (e.g., DEKK, DDKK, and/or KIH mutations) , and optional mutations silencing effector function (e.g., LALA, and/or P329A/G mutations) .

Multi-specific protein of Format I-1 contains NKp46 and target antigen-binding Fabs fused in tandem to N-terminus of one Fc chain, while IL-15 appended with a sushi domain is fused to N-terminus of the other Fc chain. Multi-specific protein of Format I-2 positions IL15 and sushi domain separately, each has its C-terminus fused to N-terminus of one Fc chain while its N-terminus fused to one of NKp46 and antigen-binding domains (Fab) , respectively. Multi-specific protein of Format I-3 comprises a sushi domain fused to N-terminus of one Fc chain and the IL15 positioned at C-terminus of a separate chain, the sushi domain and IL15 are respectively fused to C-terminus of one chain of variable domain specifically binding to antigen B, and each of IL15 and sushi domain containing amino acid substitution for interchain-di-sulfide bridge formation facilitating IL15-sushi complexing (e.g., L52C in IL15 pairing with S40C in sushi domain) ; and a Fab binding antigen A is positioned at N-termius of the other Fc chain.

Multi-specific protein of Format II has its IL15 and/or sushi domain located separately (optionally by a glycine-serine linker, such as G4S, (G4S) ₂, etc) at C-terminus of each Fc (effector function silenced) domain, forming IL15-sushi complex by non-covalent binding. Antigen binding domains (e.g., Fab or scFv) towards NKp46 and target antigen (one annotated as A, the other annotated as B, or vice versa) are fused separately to N-terminus of the two Fc chains. Sub-Formats II-1 to II-4 vary from each other by IL-15 mutations, and linkage of IL-15, as shown in Figure 5.

Multi-specific protein of Format III has its IL15 and/or sushi domain (if exists) located separately (optionally by a glycine-serine linker such as G4S, (G4S) ₂, etc. ) at C-terminus of each effector function-competent Fc chain, forming IL15-sushi complex by non-covalent binding. Antigen binding domains (e.g., Fab, scFv, or VHH) towards NKp46 and target antigen (one annotated as A, the other annotated as B, or vice versa) are fused separately to N-terminus of the two Fc chains. Format III-1 is similarly designed as Format II-2 except for having an effector function competent Fc domain; substituting the scFv domain of Format III-1 by a VHH leads to Format III-2, such scFv-VHH substitution is also contemplated for sub-Formats III-1a to III-1, as illustrated by the design of III-2a from III-1a (see Figure 6) .

Multi-specific protein of Format IV comprises two light chains and two heavy chains of an antibody comprising two Fab fragments and that specifically binds to antigen A, and fused respectively to C-terminus of two Fc chains, a binding domain (e.g. scFv, VHH) against antigen B and an IL15-sushi complex. IL15 and sushi domain are linked together by a linker from N-terminus to C-terminus and form a complex. One of A and B is NKp46, while the other is a target antigen different from NKp46. Optionally, the Fc is effector function silenced (e.g., with LALA, and/or P329A/G mutations) . Exemplary constructions of Format IV are shown in Figure 8, where IV-1, IV-2, and IV-3 vary by IL-15 mutations incorporated therein.

Multi-specific protein of Format T5 comprises, from N-terminus to C-terminus, binding domain (Fab) against antigen A, Fc domain capable of binding CD16a, a second binding domain (VH-Cκ/Vκ-CH1 complex) against antigen B and cytokine moiety, wherein T5-I to T5-III differ by the cytokine moiety therein respectively being IL15v-sushi complex, IL15v (N65A) and IL2v (R38A+F42K+C125A) . One of A and B is NKp46, while the other is a target antigen different from NKp46. IL15v/IL2v is fused to C-terminus of polypeptide chain of Vκ-CH1 by a linker (e.g., a glycine-serine linker such as G4S, (G4S) ₂, etc. ) , and sushi domain (if exists) is linked by a linker (identical to the one linking IL15 or not, e.g., a glycine-serine linker such as G4S, (G4S) ₂, etc. ) to C-terminus of VH-Cκ chain (see Figure 9) .

In a further embodiment, the present disclosure provides a multi-specific protein comprising

(i) a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide, wherein

the first polypeptide chain comprises, from N-terminus to C-terminus, VL_A-CL-VH_B-CH1-Fc;

the second polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1;

the third polypeptide chain comprises, from N-terminus to C-terminus, VL_B-CL; and

the fourth polypeptide chain comprises, from N-terminus to C-terminus, IL15Rα-linker-IL15-Fc; or

(ii) a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide, wherein

the first polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1-linker-IL15-linker-Fc;

the second polypeptide chain comprises, from N-terminus to C-terminus, VL_A-CL;

the third polypeptide chain comprises, from N-terminus to C-terminus, VH_B-CH1; and

the fourth polypeptide chain comprises, from N-terminus to C-terminus VL_B-CL-linker-IL15Rα-linker-Fc;

wherein in (i) or (ii) ,

said antigen A is NKp46 and antigen B is the target antigen, or antigen B is NKp46 and antigen A is the target antigen;

wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, for example, the Fc of IgG1, optionally the Fc of human IgG1;

the four polypeptide chains of said protein are capable of associating to form a binding protein having one first Fab specificity binding to A formed by VH_A-CH1: : VL_A-CL pairing, and one second Fab specificity binding to B formed by VH_B-CH1: : VL_B-CL pairing;

the linkers therein are independent from each other and can be the same or different;

optionally the IL15Rα is a sushi domain of said IL15Rα;

optionally, said Fc domains contain Fc-engineered mutations favoring heterodimerization; for example, KIH, DDKK/DEKK mutaion or combination thereof.

In a further embodiment, the present disclosure provides a multi-specific protein comprising:

a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide, wherein

the first polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1-Fc;

the second polypeptide chain comprises, from N-terminus to C-terminus, VL_A-CL;

the third polypeptide chain comprises, from N-terminus to C-terminus, VL_B-linker-IL15; and

the fourth polypeptide chain comprises, from N-terminus to C-terminus VH_B-linker-IL15Rα-linker-Fc;

wherein antigen A is NKp46 and antigen B is the target antigen, or B is NKp46 and A is the target antigen;

wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, for example, the Fc of IgG1, such as the Fc of human IgG1;

the four polypeptide chains are capable of associating to form a binding protein having one Fab specificity binding to A formed by VH_A-CH1: : VL_A-CL pairing, and one Fv specificity binding to B formed by VH_B: : VL_B pairing;

wherein optionally, each the IL15 and IL15Rα contain a cysteine substitution, forming an interchain-di-sulfide bridge therebetween;

optionally the IL15Rα is a sushi domain of said IL15Rα;

optionally, said IL15 is a wild type IL15 or a variant comprising one or more amino acid substitutions leading to a reduced IL-15 potency; optionally, said amino acid substitutions are selected from the group consisting of D61A, E64A, N65A, I68A, L69A, N72A, N65E, N65S, N65V and a combination thereof; optionally, said substitution are selected from N65A, L69A or N65A+L69A;

optionally, said Fc domains contain Fc-engineered mutations favoring heterodimerization; for example, KIH, DDKK/DEKK mutation or combination thereof.

a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain,

wherein the first polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1-Fc-linker-IL15Rα;

the second polypeptide chain comprises, from N-terminus to C-terminus, VL_A-CL; and

the third polypeptide chain comprises, from N-terminus to C-terminus, scFv_B-linker-Fc-linker-IL15; or scFv_B-linker-Fc-IL15;

optionally wherein, said Fc is effector function silenced, competent, or enhanced;

wherein antigen A is NKp46 and antigen B is the target antigen, or antigen B is NKp46 and antigen A is the target antigen;

wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, for example, the Fc of IgG1, such as the Fc of human IgG1; scFv is a single-chain variable fragment comprising or consisting of, from N-terminus to C-terminus, VL_B-linker-VH_B, or VH_B-linker-VL_B;

the three polypeptide chains are capable of associating to form a binding protein having one Fab specificity binding to A formed by VH_A-CH1: : VL_A-CL pairing, and one scFv specificity binding to B;

optionally the IL15Rα is a sushi domain of said IL15Rα;

the third polypeptide chain comprises, from N-terminus to C-terminus, VHH_B-linker-Fc-linker-IL15;

wherein Fc is effector function silenced or competent;

wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, for example, the Fc of IgG1 such as the Fc of human IgG1; VHH comprises or consists of a single heavy chain variable domain;

the three polypeptide chains are capable of associating to form a binding protein having one Fab specificity binding to A formed by VH_A-CH1: : VL_A-CL pairing, and one VHH specificity binding to B;

optionally the IL15Rα is a sushi domain of said IL15Rα;

In a further embodiment, the present disclosure provides a multi-specific proteins comprising

one first polypeptide chain, two second polypeptide chains, and one third polypeptide chain,

wherein the first polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1-Fc-linker-scFv_B;

the third polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1-Fc-IL15-linker-IL15Rα;

wherein Fc is effector function silenced or competent;

wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, for example, the Fc of IgG1 such as the Fc of human IgG1; scFv is a single-chain variable fragment comprising or consisting of, from N-terminus to C-terminus, VH_B-linker-VL_B or VL_B-linker-VH_B;

the four polypeptide chains are capable of associating to form a binding protein having two Fabs specificity binding to A formed by VH_A-CH1: : VL_A-CL pairing, and one scFv comprising specificity binding to B;

optionally the IL15Rα is a sushi domain of said IL15Rα;

wherein the first polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1-Fc;

the third polypeptide chain comprises, from N-terminus to C-terminus, scFv_B-linker-Fc-linker-IL15;

wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, for example, the Fc of IgG1, such as the Fc of human IgG1; scFv is a single-chain variable fragment comprising or consisting of, from N-terminus to C-terminus, VH_B-linker-VL_B or VL_B-linker-VH_B;

wherein the first polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1-Fc-VH_B-CL-linker-IL15Rα;

the second polypeptide chain comprises, from N-terminus to C-terminus, VL_A-CL-Fc; and

the third polypeptide chain comprises, from N-terminus to C-terminus, VL_B-CH1-linker-IL15;

wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, for example, the Fc of IgG1 such as the Fc of human IgG1;

the three polypeptide chains are capable of associating to form a binding protein having one Fab specificity binding to A formed by VH_A-CH1: : VL_A-CL pairing, and one Fab specificity binding to B formed by VH_B-CL: : VL_B-CH1 pairing;

optionally the IL15Rα is a sushi domain of said IL15Rα;

wherein the first polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1-Fc-VH_B-CL;

Antigen binding portion

In some embodiments, the antigen-binding portion of the multi-specific protein is aFab.

A Fab fragment suitable for use as an antigen binding portion (ABP) of the protein consists of two polypeptide chains comprising the antibody VH, CH1, VL and CL domains, wherein the VH is paired with VL and the CH1 is paired with CL to form the antigen binding portion.

In some embodiments, in a Fab, one chain comprises or consists of VH and CH1 (i.e., VH-CH1) from N-terminus to C-terminus, and the other chain comprises or consists of VL and CL (i.e., VL-CL) from N-terminus to C-terminus. In some embodiments, in the fusion protein of the present disclosure, the Fab may be linked to the N-terminus of the Fc domain of the antibody via the C-terminus of the chain comprising the VH. For example, the Fab comprises a VH-CH1 chain and a VL-CL chain and may be linked to an Fc domain via the C-terminus of CH1 of the VH-CH1 chain. In some embodiments, the linkage is a direct linkage, or through a hinge region. Herein, Fab chain comprising VH-CH1 is also referred to as a Fab heavy chain, while Fab chain comprising VL-CL is also referred to as a Fab light chain.

In some embodiments, the CH1 is a CH1 from IgG1, IgG2, IgG3, or IgG4, for example a CH1 from IgG1. In some embodiments, the CH1 comprises or consists of:

(i) an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 37,

(ii) the amino acid sequence of SEQ ID NO: 37; or

(iii) an amino acid sequence having one or more (for example not more than 10, for example, not more than 5, 4, 3, 2, 1) amino acid changes (for example amino acid substitutions, for example amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 37.

In some embodiments, the CL is a Kappa light chain constant region or a Lambda light chain constant region. In some embodiments, CL is a Kappa light chain constant region. In some embodiments, the CL comprises or consists of:

(i) an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 4,

(ii) the amino acid sequence of SEQ ID NO: 4; or

(iii) an amino acid sequence having one or more (for example not more than 10, for example not more than 5, 4, 3, 2, 1) amino acid changes (for example amino acid substitutions, for example amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the antigen-binding portion of the multi-specific protein is a Fv fragment. An Fv fragment suitable for use as an antigen binding portion of the protein consists of the antibody VH and VL domains.

In some embodiments, the antigen-binding portion of the multi-specific protein is an scFv fragment. An scFv fragment suitable for use as an antigen binding portion of the protein comprises one chain comprises or consists of VH and VL and optionally a linker. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NOs: 54-58. In certain embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 57. In some embodiments, the scFv fragment, from N-terminus to C-terminus, comprises or consists of VH-linker-VL, or VL-linker-VH. In some embodiments, the VH and VL comprises mutations to form disulfide bond. In some embodiments said scFv comprises one or more cysteine substitutions, forming a disulfide bond in the scFv chain; optionally, the VL in the scFv contains the amino acid substitution Q100C, and the VH in the scFv contains the amino acid substitution G44C (numbering according to the EU index) ..

In some embodiments, the antigen-binding portion of the multi-specific protein is an VHH. VHH generally comprises 3 CDRs and 4 FRs and generally have the following structure: FR1-CDR-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refers to Frame region 1 to 4, and CDR1 to CDR3 refers to complementarity determining regions 1-3. In some embodiments, the VHH comprises or consists of a variable region. In some embodiments, the VHH comprises an amino acid sequence of SEQ ID NO: 128.

NKp46 binding portion

In an aspect, the antigen binding region that specifically binds NKP46 (NKp46 binding portion) suitable for constituting the multi-specific protein in the present disclosure may comprise, or consist of, an anti-NKp46 full-length antibody or antigen-binding fragment thereof, as long as it can specifically bind to NKp46, the NKp46 ABP includes but not limited to, full-length antibodies, Fv, single-chain Fv (scFv) , Fab, Fab', (Fab) 2, single-domain antibodies (sdAb) , VHH, nanobody, or heavy chain antibodies that specifically bind to NKp46, etc.

In some embodiments, the NKp46-binding portion is derived from an antibody that specifically binds NKp46, e.g., NKp46-1 mab described in International PCT Publication NO. WO2022258673A1. In some embodiments, the antigen binding portion comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds NKp46, such as NKp46-1 mab described in International PCT Publication NO. WO2022258673A1. In some embodiments, the antigen-binding region comprises 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR 3, of a known antibody that specifically binds NKp46, such as NKp46-1 mab described in International PCT Publication NO. WO2022258673A1. In some embodiments, the antigen binding portion comprises 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR 3, of a known antibody that specifically binds NKp46, such as NKp46-1 mab described in International PCT Publication NO. WO2022258673A1. In some embodiments, the antigen binding portion comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of a known antibody that specifically binds NKp46, such as NKp46-1 mab described in International PCT Publication NO. WO2022258673A1. In some embodiments, the antigen binding portion comprises the heavy chain variable region and/or the light chain variable region of a known antibody that specifically binds NKp46, such as NKp46-1 mab described in International PCT Publication NO. WO2022258673A1.

In some embodiments, the antigen binding portions comprises a Fab, Fv or scFv of a known antibody that specifically binds NKp46, such as NKp46-1 mab described in International PCT Publication NO. WO2022258673A1.

In some embodiments, the NKp46-binding portion comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are HCDR1, HCDR2, and HCDR3 of a heavy chain variable region as set forth in SEQ ID NO: 60; and 3 complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein said LCDR1, LCDR2, and LCDR3 are LCDR1, LCDR2, and LCDR3 of the light chain variable region set forth in SEQ ID NO: 61.

In some aspects, the NKp46-binding portion comprises a heavy chain variable region (VH) , wherein the VH comprises or consists of

(i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 60, or

(ii) an amino acid sequence of SEQ ID NO: 60.

In some aspects, the antigen binding region that specifically binds NKP46 comprises a light chain variable region (VL) , wherein the VL

(i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 61, or

(ii) an amino acid sequence of SEQ ID NO: 61.

In some embodiments, the NKp46-binding portion comprises a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 60, and/or the VL comprises or consists of the sequence set forth in SEQ ID NO: 61.

In some embodiments, the NKp46-binding portion comprises or consists of a Fab or scFv or Fv comprising a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 60, and/or the VL comprises or consists of the sequence set forth in SEQ ID NO: 61.

In some embodiments, the NKp46-binding portion comprises an scFv; in some embodiments, said scFv chain comprises or consists of, from N-terminus to C-terminus, VL-linker-VH, or VH-linker-VL; in a particular embodiment, the linker is selected from the amino acid sequence of SEQ ID NOs: 54 to 58; in certain embodiments, said linker has the sequence of SEQ ID NO: 57. In some embodiments said scFv comprises one or more cysteine substitutions, forming a disulfide bond in the scFv chain; optionally, the VL in the scFv contains an amino acid substitution Q100C, and the VH in the scFv contains an amino acid substitution G44C (numbering according to the EU index) .

In some embodiments, wherein the scFv comprises or consists of

(i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 62, or

(ii) an amino acid sequence of SEQ ID NO: 62.

In some embodiments, the NKp46-binding portion is derived from the anti-NKp46 antibody of the present disclosure as described below, e.g., the Fab fragment of the anti-NKp46 antibody of the present disclosure as described below.

In some embodiments, the NKp46 binding portion is derived from the anti-NKp46 antibody of the present disclosure as described below, e.g., the Fv fragment of the anti-NKp46 antibody of the present disclosure as described below.

In some embodiments, the NKp46 binding portion is derived from the anti-NKp46 antibody of the present disclosure as described below, e.g., the scFv fragment of the anti-NKp46 antibody of the present disclosure as described below.

In one embodiment, the multi-specific protein as described herein comprise a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 derived from any anti-NKp46 antibody or antigen-binding fragment thereof according to the present disclosure and described below to form the NKp46 binding portion of the multi-specific protein. In some further embodiments, the multi-specific proteins as described herein comprise a VH/VL pair derived from any anti-NKp46 antibody or antigen-binding fragment thereof according to the present disclosure and described below to form the NKp46 binding region of the multi-specific binding protein.

In some embodiments, the NKp46-binding portion comprises or consists of a Fab or scFv or Fv comprising a VH and a VL, wherein the VH and VL pair as described for the anti-NKp46 antibody or antigen-binding fragment thereof according to the present disclosure and described below.

In some embodiments, the antigen binding region comprises an scFv; in some embodiments, said scFv chain comprises or consists of, from N-terminus to C-terminus, VL-linker-VH, or VH-linker-VL; in a particular embodiment, the linker is selected from the amino acid sequence of SEQ ID NOs: 54 to 58; in certain embodiments, said linker has the sequence of SEQ ID NO: 57. In some embodiments said scFv comprises one or more cysteine substitutions, forming a disulfide bond in the scFv chain; optionally, the VL in the scFv contains an amino acid substitution Q100C, and the VH in the scFv contains an amino acid substitution G44C (numbering according to the EU index) .

In some embodiments, wherein the scFv comprises or consists of

(i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 59, or

(ii) an amino acid sequence of SEQ ID NO: 59.

NKp46 antibody or antigen binding fragment

In one aspect, the present disclosure relates to a binding protein that binds to NKp46 (human NKp46) , e.g., an anti-NKp46 antibody or the antigen-binding fragment thereof.

In some embodiments, the anti-NKp46 antibody or the antigen-binding fragment thereof of the present disclosure comprises 3 complementarity determining regions from a heavy chain variable region (CDR-Hs) : CDR-H1, CDR-H2 and CDR-H3.

In some embodiments, the anti-NKp46 antibody or the antigen-binding fragment thereof of the present disclosure comprises 3 complementarity determining regions from a light chain variable region (CDR-Ls) : CDR-L1, CDR-L2 and CDR-L3.

In some embodiments, the anti-NKp46 antibody or the antigen-binding fragment thereof of the present disclosure comprises 3 complementarity determining regions from a heavy chain variable region (CDR-Hs) and 3 complementarity determining regions from a light chain variable region (CDR-Ls) .

In some aspects, the anti-NKp46 antibody or the antigen-binding fragment thereof of the present disclosure comprises a heavy chain variable region (VH) . In some aspects, the anti-NKp46 antibody or the antigen-binding fragment thereof of the present disclosure comprises a light chain variable region (VL) . In some aspects, the anti-NKp46 antibody or the antigen-binding fragment thereof of the present disclosure comprises a heavy chain variable region (VH) and a light chain variable region (VL) . In some embodiments, the heavy chain variable region comprises 3 complementarity determining regions (CDRs) from the heavy chain variable region: CDR-H1, CDR-H2 and CDR-H3. In some embodiments, the light chain variable region comprises 3 complementarity determining regions (CDRs) from the light chain variable region: CDR-L1, CDR-L2 and CDR-L3.

In some embodiments, the anti-NKp46 antibody or the antigen-binding fragment thereof of the present disclosure further comprises an antibody heavy chain constant region CH. In some embodiments, the anti-NKp46 antibody or the antigen-binding fragment thereof of the present disclosure further comprises an antibody light chain constant region CL. In some embodiments, the anti-NKp46 antibody or the antigen-binding fragment thereof of the present disclosure further comprises a heavy chain constant region CH and a light chain constant region CL.

In some embodiments, the heavy chain variable region of the present disclosure comprises or consists of

(i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to an amino acid sequence selected from SEQ ID NOs: 5, 15 and 17-23; or

(ii) an amino acid sequence selected from SEQ ID NOs: 5, 15 and 17-23; or

(iii) an amino acid sequence having one or more (preferably no more than 10, and more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, and more preferably amino acid conservative substitutions) as compared to an amino acid sequence selected from SEQ ID NOs: 5, 15 and 17-23, wherein preferably, the amino acid changes do not occur in CDRs.

In some embodiments, the light chain variable region of the present disclosure comprises or consists of

(i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to an amino acid sequence selected from SEQ ID NOs: 9, and 24-27; or

(ii) an amino acid sequence selected from SEQ ID NOs: 9, and 24-27; or

(iii) an amino acid sequence having one or more (preferably no more than 10, and more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, and more preferably amino acid conservative substitutions) as compared to an amino acid sequence selected from SEQ ID NOs: 9, and 24-27, wherein preferably, the amino acid changes do not occur in CDRs.

In some embodiments, the 3 complementarity determining regions from a heavy chain variable region (CDR-Hs) of the present disclosure, CDR-H1, CDR-H2 and CDR-H3, are selected from:

(i) three complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 contained in a VH set forth in SEQ ID NO: 5,

(ii) three complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 contained in a VH set forth in any one of SEQ ID NO: 15 and 17-23, and

(iii) compared to the sequence of any one of (i) and (ii) , a sequence comprising a total of at least one and no more than 5, 4, 3, 2, or 1 amino acid change (preferably amino acid substitution, and more preferably conservative substitution) in the three CDR-Hs,

preferably, the 3 CDR-Hs are determined according to Kabat scheme.

In some embodiments, the 3 complementarity determining regions from a light chain variable region (CDR-Ls) of the present disclosure, CDR-L1, CDR-L2, and CDR-L3, are selected from:

(i) three complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 contained in a VL set forth in any one of SEQ ID NO: 9 and 24-27; and

(ii) relative to the sequence of any one of (i) - (ii) , a sequence comprising a total of at least one and no more than 5, 4, 3, 2, or 1 amino acid change (preferably amino acid substitution, and more preferably conservative substitution) in the three CDR-Ls.

In some embodiments, the CDR-H1 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6, or the CDR-H1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, and more preferably conservative substitutions) as compared to the amino acid sequence set forth in SEQ ID NO: 6.

In some embodiments, the CDR-H2 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 7, or 16, or the CDR-H2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, and more preferably conservative substitutions) as compared to the amino acid sequence set forth in SEQ ID NO: 7, or 16.

In some embodiments, the CDR-H3 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 8, or the CDR-H3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, and more preferably conservative substitutions) as compared to the amino acid sequence set forth in SEQ ID NO: 8.

In some embodiments, the CDR-L1 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 10, or the CDR-L1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, and more preferably conservative substitutions) as compared to the amino acid sequence set forth in SEQ ID NO: 10.

In some embodiments, the CDR-L2 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 11, or the CDR-L2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, and more preferably conservative substitutions) as compared to the amino acid sequence set forth in SEQ ID NO: 11.

In some embodiments, the CDR-L3 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 12, or the CDR-L3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, and more preferably conservative substitutions) as compared to the amino acid sequence set forth in SEQ ID NO: 12.

In some embodiments, the antibody heavy chain constant region CH of the present disclosure is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably a heavy chain constant region of IgG1. In some embodiments, the antibody light chain constant region CL of the present disclosure is a lambda or kappa light chain constant region, preferably a kappa light chain constant region.

In some embodiments, the antibody light chain constant region CL of the present disclosure comprises or consists of:

(i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to an amino acid sequence of SEQ ID NO: 4;

(ii) an amino acid sequence of SEQ ID NO: 4; or

(iii) an amino acid sequence having one or more (preferably no more than 20 or 10, and more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, and more preferably amino acid conservative substitutions) as compared to an amino acid sequence of SEQ ID NO: 4.

In some embodiments, the antibody heavy chain constant region of the present disclosure comprises or consists of:

(i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to an amino acid sequence of SEQ ID NO: 3 or 38;

(ii) an amino acid sequence of SEQ ID NO: 3 or 38; or

(iii) an amino acid sequence having one or more (preferably no more than 20 or 10, and more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, and more preferably amino acid conservative substitutions) as compared to an amino acid sequence of SEQ ID NO: 3 or 38.

In some specific embodiments of the present disclosure, the anti-NKp46 antibody or the antigen-binding fragment thereof of the present disclosure comprises:

(i) three complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 contained in a VH set forth in SEQ ID NO: 5, and three complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 contained in a VL set forth in SEQ ID NO: 9;

(ii) three complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 contained in a VH set forth in any one of SEQ ID NO: 15 and 17-23, and three complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 contained in a VL set forth in any one of SEQ ID NO: 24-27;

preferably, the CDR-H and/or the CDR-L are determined according to Kabat scheme.

In some specific embodiments of the present disclosure, the anti-NKp46 antibody or the antigen-binding fragment thereof of the present disclosure comprises CDR-H1, CDR-H2 and CDR-H3 and CDR-L1, CDR-L2 and CDR-L3, wherein,

the CDR-H1 comprises or consists of the amino acid sequence of SEQ ID NO: 6; the CDR-H2 comprises or consists of the amino acid sequence of SEQ ID NO: 7 or 16; the CDR-H3 comprises or consists of the amino acid sequence of SEQ ID NO: 8; the CDR-L1 comprises or consists of the amino acid sequence of SEQ ID NO: 10; the CDR-L2 comprises or consists of the amino acid sequence of SEQ ID NO: 11; the CDR-L3 comprises or consists of the amino acid sequence of SEQ ID NO: 12.

In some specific embodiments of the present disclosure, the anti-NKp46 antibody or the antigen-binding fragment thereof of the present disclosure comprises the following pair of VH and VL:

In one embodiment of the present disclosure, the amino acid change described herein includes amino acid substitution, insertion or deletion. Preferably, the amino acid change described herein is an amino acid substitution, preferably a conservative substitution.

In a preferred embodiment, the amino acid change described herein occurs in a region outside the CDRs (e.g., in FR) . More preferably, the amino acid change described herein occurs in a region outside the heavy chain variable region and/or outside the light chain variable region. In some embodiments, the amino acid change described herein occurs in the Fc region of the antibody heavy chain constant region.

In certain embodiments, the substitution occurs in the CDRs of the antibody. Generally, the obtained variant has modifications (e.g., improvements) in certain biological properties (e.g., increased affinity) relative to the parent antibody and/or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity-matured antibodies. In some embodiments, the substitution occurs in CDR, e.g., in CDR-H2, e.g., to avoid deamination and/or heterogeneity during manufacturing. Specifically, the substitution can be an NG (Asn-Gly) to NA (Asn-Ala) mutation in VH and/or VL domains, such as in CDR-H or CDR-L, such as in CDR-H2.

In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to the substitution of an amino acid by another amino acid of the same class, e.g., the substitution of an acidic amino acid by another acidic amino acid, the substitution of a basic amino acid by another basic amino acid, or the substitution of a neutral amino acid by another neutral amino acid.

In certain embodiments, the antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites of an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

In certain embodiments, antibodies modified by cysteine engineering may need to be produced, such as “sulfo-Mab” , wherein one or more residues of the antibodies are replaced by cysteine residues.

In certain embodiments, the antibody provided herein can be further modified to comprise other non-protein portions known in the art and readily available.

In some embodiments, the anti-NKp46 antibody or the antigen-binding fragment thereof of the present disclosure has one or more of the following properties:

(i) showing the same or similar binding affinity and/or specificity for NKp46 as the antibody of the present disclosure;

(ii) inhibiting (e.g., competitively inhibiting) the binding of the antibody of the present disclosure to NKp46;

(iii) binding to the same or overlapping epitope as the antibody of the present disclosure;

(iv) competing with the antibody of the present disclosure for binding to NKp46;

(v) having one or more biological properties of the antibody of the present disclosure.

In one embodiment, the isolated anti-NKp46 antibody or antigen-binding fragment according to the present disclosure is a chimeric antibody or a humanized antibody. In some embodiments, the anti-NKp46 antibody or antigen-binding fragment is a humanized antibody.

In one embodiment, the humanized isolated anti-NKp46 antibody or antigen-binding fragment according to the present disclosure comprises one or more back mutations at positions in framework regions to improve the binding property.

In some embodiments, the VH domain of the humanized anti-NKp46 antibody or antigen-binding fragment according to the present disclosure comprises back mutations from human to residues: a Glu at position 1 (1E) , an Ile at position 2 (2I) , a Val at position 71 (71V) , and optionally one or more of a Ser at position 28 (28S) , a Ser at position 44 (44S) , an Ile at position 48 (48I) , a Lys at position (66K) , an Ala at position 67 (67A) , a Leu at position 69 (69L) , a Lys at position 73 (73K) , and an Asn at position 76 (76N) , according to Kabat numbering.

In one embodiment, the VL domain of the humanized anti-NKp46 antibody or antigen-binding fragment according to the present disclosure comprises back mutations from human to residues: a Tyr at position 71 (71Y) , and optionally one or more of a Phe at position 36 (36F) , a Trp at position 47 (47W) , and a Tyr at position 49 (49Y) , according to Kabat numbering.

In one embodiment, the isolated anti-NKp46 antibody or antigen-binding fragment according to the present disclosure is a humanized antibody, comprising back-mutated amino acid residues in the VH domain selected from the group consisting of: (i) 1E, 2I, 44S, 48I, 69L, 71V, 73K, and 76N, (ii) 1E, 2I, and 71V, (iii) 1E, 2I, 71V, and 73K, (iv) 1E, 2I, 48I, 66K, 67A, 71V, and 73K, (v) 1E, 2I, 28S, and 71V, and (vi) 1E, 2I, 28S, 48I, 66K, 67A, 71V, and 73K, all according to Kabat numbering; and/or back-mutated amino acid residues in the VL domain selected from the group consisting of: (i) 36F, 47W, 49Y and 71Y; (ii) 71Y, and (iii) 47W, and 71Y, all according to Kabat numbering.

In one embodiment, the isolated anti-NKp46 antibody or antigen-binding fragment according to the present disclosure is a humanized antibody, comprising amino acid residues 1E, 2I, 48I, 66K, 67A, 71V, and 73K in the VH domain, and amino acid residue 71Y in the VL domain, according to Kabat numbering.

In some embodiments, the VH domain of the humanized anti-NKp46 antibody or antigen-binding fragment according to the present disclosure comprises back mutations from human to residues: a Arg at position 94 (94R) , and optionally one or more of a Ile at position 2 (2E) , a Ser at position 28 (28S) , a Lys at position 38 (38K) , a Ser at position 44 (44S) , an Ile at position 48 (48I) , a Lys at position 66 (66K) , an Ala at position 67 (67A) , an Ala at position 69 (69A) , a Leu at position 69 (69L) , a Val at position 71 (71V) , a Ser at position 73 (73K) , and an Asn at position 76 (76N) , according to Kabat numbering. In one embodiment, the VL domain of the humanized anti-NKp46 antibody or antigen-binding fragment according to the present disclosure comprises back mutations from human to residues: a Tyr at position 49 (49Y) , a Tyr at position 71 (71Y) , and optionally one or more of a Phe at position 36 (36F) , a Trp at position 47 (47W) , and a Ser at position 70 (70S) , according to Kabat numbering.

In one embodiment, the isolated anti-NKp46 antibody or antigen-binding fragment according to the present disclosure is a humanized antibody, comprising back-mutated amino acid residues in the VH domain selected from the group consisting of: (i) 94R, (ii) 71V, 73K and 94R, (iii) 66K, 67A, 71V, 73K and 94R, (iv) 48I, 66K, 67A, 69L, 71V, 73K and 94R, (v) 2I, 28S, 48I, 66K, 67A, 71V, 73K, and 94R, and (vi) 2I, 28S, 38K, 44S, 48I, 66K, 67A, 68A, 69L, 71V, 73K, 76N, and 94R, all according to Kabat numbering; and/or back-mutated amino acid residues in the VL domain selected from the group consisting of: (i) 49Y and 71Y; (ii) 36F, 49Y, 70S, and 71Y, and (iii) 36F, 47W, 49Y, 70S, and 71Y, all according to Kabat numbering.

In one embodiment, the isolated anti-NKp46 antibody or antigen-binding fragment according to the present disclosure is a humanized antibody, comprising amino acid residues 66K, 67A, 71V, 73K, and 94R in the VH domain, and amino acid residue 36F, 49Y, 70S, and 71Y in the VL domain, according to Kabat numbering.

In a further embodiment, the isolated anti-NKp46 antibody or antigen-binding fragment according to the present disclosure further comprises DG (Asp-Gly) to DA (Asp-Ala) mutations in the VH domain according to Kabat numbering. In some embodiments of an anti-NKp46 antibody or antigen-binding fragment according to the present disclosure, the antibody or antigen-binding fragment comprises an Fc region, which may be a native or a variant Fc region. In particular embodiments, the Fc region is a human Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. Depending on the utility of the antibody, it may be desirable to use a variant Fc region to change (for example, reduce or eliminate) at least one effector function, for example, ADCC and/or CDC. In some embodiments, the present disclosure provides an anti-NKp46 antibody or antigen-binding fragment comprising an Fc region with one or more mutation to change at least one effector function, for example, L234A and L235A (abbreviated as LALA mutation hereafter) or P329A/G.

In some embodiments, antigen-binding fragments of an anti-NKp46 antibody according to the present disclosure may be for example, Fv, Fab, Fab’, Fab’-SH, F (ab’) 2; diabodies; linear antibodies; or single-chain antibody molecules (e.g., scFv) .

In one embodiment, an anti-NKp46 antibody described herein or an antigen-binding fragment thereof binds to the NKp46 extracellular domain or a portion thereof. In some embodiments, the NKp46 extracellular domain comprises the amino acid squence 22-258 of the human NKp46 protein under UniProt Identifier O76036.1, or the amino acid sequence of SEQ ID NO: 39, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identity therewith.

In one embodiment, the anti-NKp46 antibodies and an antigen-binding fragment thereof has one or more of the following activities:

(i) binding to the human NKp46 with high affinity;

(ii) binding to the human NKp46 expressed in the cell surface;

(iii) showing positive binding activity to humanNKp46, cynoNKp46, but negative binding activity to mouseNKp46; and/or

(iv) activating NK cells, e.g., upregulating membrane CD107a expression on NK cells.

IL15 and IL15R

In an aspect, the multi-specific protein comprises IL15-IL15Rα complex. In some embodiments, the IL15Rα in the complex is a sushi domain of the IL15Rα and the complex is a IL15-sushi domain complex.

The IL15-IL15Rα domain complex generally comprises IL-15 protein and the IL15Rα. This complex can be used in three different formats. In one embodiment, the IL-15 protein and the IL-15Rα are non-covalently attached, and self-assembled through regular ligand-ligand interactions. It can be either or both of the IL-15 domain and the IL15Rα domain that is covalently linked to the Fc domain (optionally using a linker) .

Alternatively, the IL15Rα domain and the IL-15 domain can be covalently attached using a linker. For example, the IL15Rα domain is covalently linked to the Fc domain either at N-terminus or the C-terminus, or the IL-15 protein is covalently linked to the Fc domain either at N-terminus or the C-terminus.

Alternatively, each of the IL-15 and IL15Rα domains can be engineered to contain a cysteine amino acid, leading to an interchain-disulfide bond to form a complex, with either the IL-15 domain or the IL15Rα domain being covalently attached (optionally using a linker) to the Fc domain.

The IL15-sushi domain complex generally comprises IL-15 protein and the sushi domain of IL-15Rα. This complex can be used in three different formats. In one embodiment, the IL-15 protein and the IL15Rα-sushi are non-covalently attached, and self-assembled through regular ligand-ligand interactions. It can be either or both of the IL-15 domain and the sushi domain that is covalently linked to the Fc domain (optionally using a linker) .

Alternatively, the sushi domain and the IL-15 domain can be covalently attached using a linker. For example, the sushi domain is covalently linked to the Fc domain either at N-terminus or the C-terminus, or the IL-15 protein is covalently linked to the Fc domain either at N-terminus or the C-terminus.

Alternatively, each of the IL-15 and sushi domains can be engineered to contain a cysteine amino acid, leading to an interchain-disulfide bond to form a complex, with either the IL-15 domain or the sushi domain being covalently attached (optionally using a linker) to the Fc domain.

In some embodiments, the IL-15 protein is a human IL-15 protein or variant thereof. In some embodiments, the human IL-15 protein has the amino acid sequence set forth in NCBI Ref. Seq. No. NP_000576.1 or SEQ ID NO: 40. In some embodiments, the coding sequence of human IL-15 is set forth in NCBI Ref. Seq. No. NM_000585. An exemplary IL-15 protein used herein can have the amino acid sequence of SEQ ID NO: 41 (mature IL-15) or amino acids 49-162 of SEQ ID NO: 41. In some embodiments, the IL-15 protein has at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 41. In some embodiments, the IL-15 protein comprises or consists of the amino acid sequence of SEQ ID NO: 41 and one or more amino acid mutations such as substitutions.

The amino acid substitution (s) may be isosteric substitutions at the IL-15: IL-20 and IL-15: common gamma chain interface. In some embodiments, the human IL-15 protein such as the human mature IL-15 protein has one or more amino acid substitutions selected from the group consisting of D61A, E64A, N65A, I68A, L69A, N72A, N65E, N65S, N65V, and any combination thereof. In some embodiments, the human IL-15 protein of the multi-specific protein has the amino acid sequence of SEQ ID NO: 41 and amino acid substitutions N65A, L69A or N65A+L69A.

In some embodiments, the human IL-15 protein of the multi-specific protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to anyone of SEQ ID NOs: 42 to 48. In some embodiments, the human IL-15 protein of the multi-specific protein comprises or consists of the amino acid as set forth in anyone or SEQ ID NOs: 42 to 48.

In some embodiments, the human IL-15 protein of the multi-specific protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 41, 44, 46 or 48 including N65A, L69A or N65A+L69A substitutions, wherein the amino acid position is relative to SEQ ID NO: 41.

In some embodiments, the human IL-15 protein of the multi-specific protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 44 and comprises N65A substitutions.

In some embodiments, the human IL-15 protein of the multi-specific protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 46 and comprises L69A substitutions.

In some embodiments, the human IL-15 protein of the multi-specific protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 48 and comprises N65A and L69A substitutions.

In some embodiments, the human IL-15 protein, such as a human mature IL-15 protein of the multi-specific protein comprises or consist of the amino acid sequence of SEQ ID NO: 41, 44, 46 or 48.

In some embodiments, the human IL-15 variant protein has one or more amino acid mutations (e.g., substitutions, insertions and/or deletions) . In some instances, the mutation introduces a cysteine residue that can form a disulfide bond with the sushi domain of the human IL-15 receptor alpha (IL-15Rα) protein. For example, in some embodiments, the IL-15 protein comprises L52C substitutions. In some embodiments, the IL-15 comprises or consists of SEQ ID NO: 51. In some embodiments, the IL-15 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 40 and comprises L52C substitutions.

In some embodiments, the human IL-15 receptor alpha (IL-15Rα) protein has the amino acid sequence set forth in NCBI Ref. Seq. No. NP_002180.1 or SEQ ID NO: 49. In some embodiments, the coding sequence of human IL-15Rα is set forth in NCBI Ref. Seq. No. NM_002189.3. An exemplary IL-15Rα protein herein can comprise or consist of the sushi domain of SEQ ID NO: 49 (e.g., amino acids 31-95 of SEQ ID NO: 49) , or the amino acid sequence of SEQ ID NO: 49.

In some embodiments, the sushi domain comprises or consists of the amino acid sequence of SEQ ID NO: 50, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 50.

In some embodiments, the sushi domain can have 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid mutations (e.g., substitutions, insertions and/or deletions) compared to the amino acid of SEQ ID NO: 50.

In some instances, the mutation introduces a cysteine residue that can form a disulfide bond with the sushi domain of the human IL-15 receptor alpha (IL-15Rα) protein. For example, in some embodiments, the sushi domain comprises S40C substitutions. In some embodiments, the sushi domain comprises or consists of SEQ ID NO: 52. In some embodiments, the sushi domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 52 and comprises S40C substitutions. In some embodiments, the sushi domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 50 and comprises S40C substitutions.

Domain Linkers

In some embodiments, regions or portions or domains contained in the portions or regions of the multispecic protein, such as the NKp46 binding portion, the target antigen binding portion, the IL-15-sushi complex and/or the Fc domain are attached together via a linker. Optionally, the regions are fused without a linker.

In some embodiments, the IL-15 protein is attached to an Fc domain via a linker. In certain embodiments, the IL-15 protein is attached to an Fc domain directly, such as without a linker. In certain embodiments, the IL-15 protein is attached to an Fc domain via a hinge region or a fragment thereof. In some embodiments, the sushi domain is attached to an Fc domain via a linker. In other embodiments, the sushi domain is attached to an Fc domain directly, such as without a linker. In particular embodiments, the sushi domain is attached to an Fc domain via a hinge region or a fragment thereof.

In some embodiments, the linker is a “peptide linker” , used to link any two regions of the multi-specific protein as outlined herein together. While any suitable linker can be used, in some embodiments, the linker has less than 10 amino acids, e.g., less than 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid (s) .

In some eombodiments, a glycine-serine polymer is used, including for example (GS) n, (GSGGS) n , (GGGGS) n, (GGGS) n, (GSSS) n or (GSSS) nGS, where n is an integer of 1-10 (and generally from 0 to 1 to 2 to 3 to 4 to 5 to 6) as well as any peptide sequence that allows for recombinant attachment of the two domains with sufficient length and flexibility to allow each domain to retain its biological function.

In certain embodiments, useful linkers include (GGGGS) ₁ (SEQ ID NO: 54) , (GGGGS) ₂ (SEQ ID NO: 55) , (GGGGS) ₃ (SEQ ID NO: 56) , (GGGGS) ₄ (SEQ ID NO: 57) or (GGGGS) ₆ (SEQ ID NO: 58) , GSSSS, (GSSSS) ₂, (GSSSS) ₃, or (GSSSS) ₃GS.

Fc regions

In some embodiments, the multi-specific protein comprises Fc region (s) , e.g., Fc dimer.

Fc fragments suitable for use in the multi-specific proteins of the present disclosure may be any antibody Fc region. Fc regions can include native sequence Fc regions and variant Fc regions. Native sequence Fc domains encompass various immunoglobulin Fc sequences that occur in nature, such as the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389/fimmu. 2014.00520. ) . For example, the Fc region of an antibody of the present disclosure may comprise two or three constant domains, i.e., a CH2 domain, a CH3 domain, and an optional CH4 domain. In some embodiments, the antibody Fc region may also carry an IgG hinge region or a portion of an IgG hinge region at the N-terminus, e.g., an IgG1 hinge region or a portion of an IgG1 hinge region.

Specifically, the Fc region of the protein of the present disclosure comprises from N-terminal to C-terminal: CH2-CH3, or from N-terminal to C-terminal: hinge region-CH2-CH3. In some embodiments, the Fc region is a human Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. In some embodiments, the Fc region suitable for use in the fusion protein of the present disclosure is a human IgG Fc, e.g., human IgGl Fc, human IgG2 Fc, or human IgG4 Fc.

In one embodiment, the Fc region comprises or consists of the amino acid sequence SEQ ID NO: 28 or an amino acid sequence having at least 90%identity thereto, e.g., 95%, 96%, 97%, 99%or more identity.

The Fc region of the protein of the present disclosure can be further mutated for desired property by Fc engineering approaches well known in the art (for example, Wang et al., Protein and Cell, 2018; 9 (1) : 63–73) .

The Fc region of the fusion protein of the present disclosure can be mutated to obtain desired properties. Mutations to the Fc region are known in the art.

In one embodiment, the Fc region is modified for the properties of the effector function of the Fc region (e.g., the complement activation function of the Fc region) . In one embodiment, the effector function has been reduced or eliminated relative to the wild-type isotype Fc region. In one embodiment, effector function is reduced or eliminated by a method selected from using Fc isotypes that naturally have reduced or eliminated effector function, and Fc region modification.

In one embodiment, the Fc region has reduced or silenced effector function mediated by the Fc region, e.g., reduced or eliminated ADCC or ADCP or CDC effector function, e.g., comprises mutations to achieve the above functions.

As will be understood by those of skill in the art, depending on the intended use, the protein molecules of the present disclosure may also comprise modifications in the Fc domain that alter the binding affinity for one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, in particular a human Fcγ receptor, optionally hFcγRIII, such as CD16a. In some embodiments, the Fc region comprises a mutation that reduces binding to Fcγ receptors. For example, in some embodiments, the Fc region used in the present disclosure has one or more of the L234A/L235A mutation (LALA mutation) and/or P329A/G mutation that reduces binding to Fcγ receptors. While in some embodiments, the Fc region used in the present disclosure carries mutations S239D+A330L+I332E (DLE mutation) to enhance Fc function of binding Fcγ receptors. In yet another embodiment, the Fc fragment may have a mutation that results in increased serum half-life, e.g., a mutation that improves the binding of the Fc fragment to FcRn. In some embodiments, the Fc region comprises a YTE mutation (M252Y/S254T/T256E) that improves the binding of the Fc fragment to FcRn. In some embodiments, the Fc region comprises a LALA mutation and/or a YTE mutation.

In some embodiments, once the two chains for dimerization are different, the Fc regions can be different and are capable of dimerizing to form a heterodimeric Fc scaffold.

As understood by those skilled in the art, to facilitate formation of the two chains as heterodimers, the Fc region comprised by the two chains may comprise mutations that facilitate heterodimerization of a first Fc region with a second Fc region. In one embodiment, mutations are introduced in the CH3 regions of both Fc regions.

Methods for promoting heterodimerization of Fc regions are known in the art. For example, the CH3 region of the first Fc region and the CH3 region of the second Fc region are engineered in a complementary manner such that each CH3 region (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with other CH3 regions that are complementarily engineered (such that the CH3 regions of the first and second Fc regions heterodimerize and no homodimers are formed between the two first CH3 regions or the two second CH3 regions) .

For example, the respective Knob mutations and Hole mutations are introduced in the first Fc region and the second Fc region based on the Knob-in-Hole ( "KIH” ) technology. See, e.g., US 5,731,168; US 7,695,936; Ridgway et al, Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001) .

In a particular embodiment, in the CH3 region of an Fc region, the threonine residue at position 366 is replaced with a tyrosine residue (T366Y) ; or the threonine residue at position 366 is replaced with a tryptophan residue (T366W) ; in the CH3 region of the other Fc region, the tyrosine residue at position 407 was replaced with a Threonine residue (Y407T) ; or, the threonine residue at position 366 was replaced with a serine residue (T366S) , the Leucine residue at position 368 is replaced with a alanine residue (L368A) and the tyrosine residue at position 407 was replaced with a valine residue (Y407V) (numbering according to the EU index) .

In a particular embodiment, each Fc region of the Fc scaffold further comprises mutations to form non-natural disulfide bond. For example, in one Fc region, the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine residue) ; and in the other Fc region, the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering according to the EU index) .

In yet another embodiment, in the CH3 region of an Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) , and the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine residue) ; whereas in the CH3 region of the other Fc region, the threonine residue at position 366 is replaced with a serine residue (T366S) , the leucine residue at position 368 is replaced with an alanine residue (L368A) , and the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (numbering according to the EU index) , optionally the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering according to the EU index) . In a particular embodiment, one Fc region comprises the amino acid substitutions S354C and T366W and the other Fc region comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the EU index) . As used herein, the mutation combination mentioned above is typically denoted as “KIH” mutation. In some embodiments, the Fc region comprises a mutation combination of DLE and/or KIH.

In a further embodiment, the Fc heterodimer formation can be further enhanced through electrostatic steering effects. For example, in one Fc region, K392D and/or K409D are introduced, and E356K and/or D399K are introduced in the other Fc region. As used herein, the mutation combination used therein is typically denoted as “DDKK” mutation.

In a particular embodiment, one Fc region comprises the amino acid substitutions S354C, T366W, K409D and K392D and the other Fc region comprises the amino acid substitutions Y349C, T366S, L368A, Y407V, D399K and E356K (numbering according to the EU index) .

Other approaches for promoting the heterodimerization was published by de Kruif and colleagues [e, N.C.; Hendriks, L.J.A.; Poirier, E.; Arvinte, T.; Gros, P.; Bakker, A.B.; de Kruif, J. A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1. J. Biol. Chem. 2017, 292, 14706–14717] . For this, double mutations are incorporated into two antibody heavy chains. The “DEKK” mutation pairs consist of substitutions L351D and L368E in one Fc chain, whereas the other Fc chain harbors L351K and T366K exchanges. The formation of salt bridges between lysine residues as well as native and substituted amino acid residues on the opposite CH3 stabilize the DEKK heterodimer.

Further available approaches and the mutation on Fc regions can be found in Antibodies 2018, 7 (3) , 28; https: //doi. org/10.3390/antib7030028.

In a particular embodiment, one Fc region comprises the amino acid substitutions LALA, L351K and T366K (LALA-KK) ; and the other Fc region comprises the amino acid substitutions LALA (L234A/L235A) , L351D and L368E (LALA-DE) , (numbering according to the EU index) .

Thus, in a particular embodiment, the molecule of the present disclosure comprises two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29, while the other Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30.

Thus, in a particular embodiment, the molecule of the present disclosure comprise two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 29, while the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 30.

Thus, in a particular embodiment, the molecule of the present disclosure comprise two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 29 and comprises the mutations LALA (L234A/L235A) , L351D and L368E, while the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 30 and comprises the mutations LALA (L234A/L235A) , L351K and T366K.

In a particular embodiment, one Fc region comprises the amino acid substitutions Y349C + T366S + L368A + Y407V; and the other Fc region comprises the amino acid substitutions S354C + T366W (numbering according to the EU index) .

Thus, in a particular embodiment, the molecule of the present disclosure comprises two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31, while the other Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32.

Thus, in a particular embodiment, the molecule of the present disclosure comprise two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 31, while the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 32.

Thus, in a particular embodiment, the molecule of the present disclosure comprise two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 31 and comprises the mutations Y349C + T366S + L368A + Y407V, while the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 32 and comprises the mutations S354C + T366W.

In a particular embodiment, one Fc region comprises the amino acid substitutions LALA (L234A/L235A) , Y349C + T366S + L368A + Y407V, and D399K + E356K; and the other Fc region comprises the amino acid substitutions LALA (L234A/L235A) , S354C + T366W, and K409D + K392D (numbering according to the EU index) .

Thus, in a particular embodiment, the molecule of the present disclosure comprises two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 33, while the other Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34.

Thus, in a particular embodiment, the molecule of the present disclosure comprise two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 33, while the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 34.

Thus, in a particular embodiment, the molecule of the present disclosure comprise two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 33 and comprises the mutations LALA (L234A/L235A) , Y349C + T366S + L368A + Y407V, and D399K + E356K, while the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 34 and comprises the mutations LALA (L234A/L235A) , S354C + T366W, and K409D +K392D.

In a particular embodiment, one Fc region comprises the amino acid substitutions DLE and Y349C + T366S + L368A + Y407V; and the other Fc region comprises the amino acid substitutions DLE and S354C + T366W (numbering according to the EU index) .

Thus, in a particular embodiment, the molecule of the present disclosure comprises two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35, while the other Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 36.

Thus, in a particular embodiment, the molecule of the present disclosure comprise two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 35, while the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 36.

Thus, in a particular embodiment, the molecule of the present disclosure comprise two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 35 and comprises the mutations DLE and Y349C + T366S + L368A + Y407V, while the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 36 and comprises the mutations DLE and S354C + T366W.

Target antigen binding portion

In an aspect, the second binding portion suitable for constituting the multi-specific protein in the present disclosure may comprise, or consist of, a full-length antibody or antigen-binding fragment thereof, as long as it can specifically bind to a scond antigen, including but not limited to, full-length antibodies, single-chain Fv, Fab, Fab', (Fab) 2, single-domain antibodies, VHH or heavy chain antibodies that specifically bind to the target antigen, etc.

In some embodiments, the target antigen can be a cancer antigen. In some embodiments, the cancer antigen is a tumor-associated antigen. In some embodiments, the cancer antigen is a check point molecule. In some embodiments, the target antigen is selected from Her2, Trop2, LIV-1, ALPP, CD20, CD123, and CD228.

In some embodiments, the second binding region is derived from an antibody specifically binds to a target antigen, e.g., the Fab fragment, Fv fragment or scFv fragment of the antibody.

In some embodiments, the target antigen binding portion comprises 1, 2, 3, 4, 5, or 6 CDRs of an antibody that specifically binds the target antigen.

In some embodiments, the target antigen binding portion comprises 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of an antibody that specifically binds a target antigen.

In some embodiments, the target antigen binding portion comprises 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of an antibody that specifically binds a target antigen.

In some embodiments, the target antigen binding portion comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of an antibody that specifically binds a target antigen.

In some embodiments, the target antigen binding portion comprises the heavy chain variable region and the light chain variable region of an antibody that specifically binds a target antigen.

In one embodiment, the multi-specific protein as described herein comprise a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 derived from any antibody or antigen-binding fragment thereof against the target antigen to form the second binding portion of the multi-specific protein. In some further embodiments, the multi-specific proteins as described herein comprise a VH/VL pair derived from any antibody or antigen-binding fragment thereof against the target antigen to form the second binding portion of the multi-specific protein.

In some embodiments, the target antigen binding portions comprises a Fab of an antibody that specifically binds a target antigen.

In some embodiments, the target antigen binding portions comprises an Fv of an antibody that specifically binds a target antigen.

In some embodiments, the target antigen binding portions comprises an scFv of an antibody that specifically binds a target antigen. In some embodiments, said scFv chain is, from N-terminus to C-terminus, VL-linker-VH, or VH-linker-VL; in a particular embodiment, the linker is selected from the amino acid sequence of SEQ ID NOs: 55 to 58; in certain embodiments, said linker has the sequence of SEQ ID NO: 57. In some embodiments said scFv comprises one or more cysteine substitutions, forming a disulfide bond in the scFv chain; optionally, the VL in the scFv contains the amino acid substitution Q100C, and the VH in the scFv contains the amino acid substitution G44C (numbering according to the EU index) .

In some embodiments, the target antigen binding portions comprises a VHH of an antibody that specifically binds a target antigen.

In some embodiments, the target antigen binding portion specifically binds to Her2.

In some embodiments, the antigen binding portion is derived from an antibody that specifically binds Her2, e.g., trastuzumab. In some embodiments, the antigen binding portion comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds Her2, such as trastuzumab. In some embodiments, the antigen-binding region comprises 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR 3, of a known antibody that specifically binds Her2, such as trastuzumab. In some embodiments, the antigen binding portion comprises 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR 3, of a known antibody that specifically binds Her2, such as trastuzumab. In some embodiments, the antigen binding portion comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of a known antibody that specifically binds Her2, such as trastuzumab. In some embodiments, the antigen binding portion comprises the heavy chain variable region and/or the light chain variable region of a known antibody that specifically binds Her2, such as trastuzumab. In some embodiments, the antigen binding portions comprises a Fab, scFv or VHH of a known antibody that specifically binds Her2, such as trastuzumab.

In some embodiments, the antigen binding region that specifically binds HER2 comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are HCDR1, HCDR2, and HCDR3 of a heavy chain variable region as set forth in SEQ ID NO: 65; and 3 complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein said LCDR1, LCDR2, and LCDR3 are LCDR1, LCDR2, and LCDR3 of the light chain variable region set forth in SEQ ID NO: 66.

In some aspects, the antigen binding region that specifically binds HER2 comprises a heavy chain variable region (VH) , wherein the VH comprises or consists of

(i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 65, or

(ii) an amino acid sequence of SEQ ID NO: 65.

In some aspects, the antigen binding region that specifically binds HER2 comprises a light chain variable region (VL) , wherein the VL comprises or consists of

(i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 66, or

(ii) an amino acid sequence of SEQ ID NO: 66.

In some embodiments, the antigen binding region that specifically binds HER2 comprises a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 65, and/or the VL comprises or consists of the sequence set forth in SEQ ID NO: 66.

In some embodiments, the antigen binding region comprises or consists of a Fab or scFv or Fv comprising a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 65, and/or the VL comprises or consists of the sequence set forth in SEQ ID NO: 66.

In some embodiments, the antigen binding region comprises an scFv, wherein the scFv comprises or consists of

(i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 71, or

(ii) an amino acid sequence of SEQ ID NO: 71.

In some embodiments, the target antigen binding portion specifically binds to CD20.

In some embodiments, the antigen binding portion is derived from an antibody that specifically binds CD20, e.g., CD20-2 mab mentioned in WO2022258673A1. In some embodiments, the antigen binding portion comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds CD20, such as CD20-2 mab mentioned in WO2022258673A1. In some embodiments, the antigen-binding region comprises 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR 3, of a known antibody that specifically binds CD20, such as CD20-2 mab mentioned in WO2022258673A1. In some embodiments, the antigen binding portion comprises 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR 3, of a known antibody that specifically binds CD20, such as CD20-2 mab mentioned in WO2022258673A1. In some embodiments, the antigen binding portion comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of a known antibody that specifically binds CD20, such as CD20-2 mab mentioned in WO2022258673A1. In some embodiments, the antigen binding portion comprises the heavy chain variable region and/or the light chain variable region of a known antibody that specifically binds CD20, such as CD20-2 mab mentioned in WO2022258673A1. In some embodiments, the antigen binding portions comprises a Fab, Fv or scFv of a known antibody that specifically binds CD20, such as CD20-2 mab mentioned in WO2022258673A1.

In some embodiments, the antigen binding region that specifically binds CD20 comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are HCDR1, HCDR2, and HCDR3 of a heavy chain variable region as set forth in SEQ ID NO: 63; and 3 complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein said LCDR1, LCDR2, and LCDR3 are LCDR1, LCDR2, and LCDR3 of the light chain variable region set forth in SEQ ID NO: 64.

In some aspects, the antigen binding region that specifically binds CD20 comprises a heavy chain variable region (VH) , wherein the VH

(i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 63, or

(ii) comprises or consists of an amino acid sequence of SEQ ID NO: 63.

In some aspects, the antigen binding region that specifically binds CD20 comprises a light chain variable region (VL) , wherein the VL

(i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 64, or

(ii) comprises or consists of an amino acid sequence of SEQ ID NO: 64.

In some embodiments, the antigen binding region that specifically binds CD20 comprises a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 63, and/or the VL comprises or consists of the sequence set forth in SEQ ID NO: 64.

In some embodiments, the antigen binding region comprises or consists of a Fab or scFv or Fv comprising a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 63, and/or the VL comprises or consists of the sequence set forth in SEQ ID NO: 64.

In some embodiments, the target antigen binding portion specifically binds to LIV-1.

In some embodiments, the antigen binding portion is derived from an antibody that specifically binds LIV-1, e.g., ladiratuzumab. In some embodiments, the antigen binding portion comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds LIV-1, such as ladiratuzumab. In some embodiments, the antigen-binding region comprises 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR 3, of a known antibody that specifically binds LIV-1, such as ladiratuzumab. In some embodiments, the antigen binding portion comprises 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR 3, of a known antibody that specifically binds LIV-1, such as ladiratuzumab. In some embodiments, the antigen binding portion comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of a known antibody that specifically binds LIV-1, such as ladiratuzumab. In some embodiments, the antigen binding portion comprises the heavy chain variable region and/or the light chain variable region of a known antibody that specifically binds LIV-1, such as ladiratuzumab. In some embodiments, the antigen binding portions comprises a Fab, Fv or scFv of a known antibody that specifically binds LIV-1, such as ladiratuzumab.

In some embodiments, the antigen binding region y that specifically binds LIV-1 comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are HCDR1, HCDR2, and HCDR3 of a heavy chain variable region as set forth in SEQ ID NO: 67; and 3 complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein said LCDR1, LCDR2, and LCDR3 are LCDR1, LCDR2, and LCDR3 of the light chain variable region set forth in SEQ ID NO: 68.

In some aspects, the antigen binding region that specifically binds LIV-1 comprises a heavy chain variable region (VH) , wherein the VH

(i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 67, or

(ii) comprises or consists of an amino acid sequence of SEQ ID NO: 67.

In some aspects, the antigen binding region that specifically binds LIV-1 comprises a light chain variable region (VL) , wherein the VL

(i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 68, or

(ii) comprises or consists of an amino acid sequence of SEQ ID NO: 68.

In some embodiments, the antigen binding region that specifically binds LIV-1 comprises a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 67, and/or the VL comprises or consists of the sequence set forth in SEQ ID NO: 68.

In some embodiments, the antigen binding region comprises or consists of a Fab or scFv or Fv comprising a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 67, and/or the VL comprises or consists of the sequence set forth in SEQ ID NO: 68.

In some embodiments, the target antigen binding portion specifically binds to CD228.

In some embodiments, the antigen binding portion is derived from an antibody that specifically binds CD228, e.g., CD228 mab clone#SC57.10 mentioned in US 10, 428, 156 B2. In some embodiments, the antigen binding portion comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds CD228, such as CD228 mab clone#SC57.10 mentioned in US 10, 428, 156 B2. In some embodiments, the antigen-binding region comprises 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR 3, of a known antibody that specifically binds CD228, such as CD228 mab clone#SC57.10 mentioned in US 10, 428, 156 B2. In some embodiments, the antigen binding portion comprises 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR 3, of a known antibody that specifically binds CD228, such as CD228 mab clone#SC57.10 mentioned in US 10, 428, 156 B2. In some embodiments, the antigen binding portion comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of a known antibody that specifically binds CD228, such as CD228 mab clone#SC57.10 mentioned in US 10, 428, 156 B2. In some embodiments, the antigen binding portion comprises the heavy chain variable region and/or the light chain variable region of a known antibody that specifically binds CD228, such as CD228 mab clone#SC57.10 mentioned in US 10, 428, 156 B2. In some embodiments, the antigen binding portions comprises a Fab, Fv or scFv of a known antibody that specifically binds CD228, such as CD228 mab clone#SC57.10 mentioned in US 10, 428, 156 B2.

In some embodiments, the antigen binding region that specifically binds CD228 comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are HCDR1, HCDR2, and HCDR3 of a heavy chain variable region as set forth in SEQ ID NO: 69; and 3 complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein said LCDR1, LCDR2, and LCDR3 are LCDR1, LCDR2, and LCDR3 of the light chain variable region set forth in SEQ ID NO: 70.

In some aspects, the antigen binding region that specifically binds CD228 comprises a heavy chain variable region (VH) , wherein the VH

(i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 69, or

(ii) comprises or consists of an amino acid sequence of SEQ ID NO: 69.

In some aspects, the antigen binding region that specifically binds CD228 comprises a light chain variable region (VL) , wherein the VL

(i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 70, or

(ii) comprises or consists of an amino acid sequence of SEQ ID NO: 70.

In some embodiments, the antigen binding region that specifically binds CD228 comprises a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 69, and/or the VL comprises or consists of the sequence set forth in SEQ ID NO: 70.

In some embodiments, the antigen binding region comprises or consists of a Fab or scFv or Fv comprising a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 69, and/or the VL comprises or consists of the sequence set forth in SEQ ID NO: 70.

In some embodiments, the target antigen binding portion specifically binds to CD123.

In some embodiments, the antigen binding portion is derived from an antibody that specifically binds CD123, e.g., CD123 mab talacotuzumab. In some embodiments, the antigen binding portion comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds CD123, such as talacotuzumab. In some embodiments, the antigen-binding region comprises 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR 3, of a known antibody that specifically binds CD123, such as talacotuzumab. In some embodiments, the antigen binding portion comprises 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR 3, of a known antibody that specifically binds to CD123, such as talacotuzumab. In some embodiments, the antigen binding portion comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of a known antibody that specifically binds CD123, such as talacotuzumab. In some embodiments, the antigen binding portion comprises the heavy chain variable region and/or the light chain variable region of a known antibody that specifically binds CD123, such as talacotuzumab. In some embodiments, the antigen binding portions comprises a Fab, Fv or scFv of a known antibody that specifically binds CD123, such as talacotuzumab.

In some embodiments, the antigen binding region that specifically binds CD123 comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are HCDR1, HCDR2, and HCDR3 of a heavy chain variable region as set forth in SEQ ID NO: 145; and 3 complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein said LCDR1, LCDR2, and LCDR3 are LCDR1, LCDR2, and LCDR3 of the light chain variable region set forth in SEQ ID NO: 146.

In some aspects, the antigen binding region that specifically binds CD123 comprises a heavy chain variable region (VH) , wherein the VH

(i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 145, or

(ii) comprises or consists of an amino acid sequence of SEQ ID NO: 145.

In some aspects, the antigen binding region that specifically binds CD123 comprises a light chain variable region (VL) , wherein the VL

(i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 146, or

(ii) comprises or consists of an amino acid sequence of SEQ ID NO: 146.

In some embodiments, the antigen binding region that specifically binds CD123 comprises a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 145, and/or the VL comprises or consists of the sequence set forth in SEQ ID NO: 146.

In some embodiments, the antigen binding region comprises or consists of a Fab or scFv or Fv comprising a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 145, and/or the VL comprises or consists of the sequence set forth in SEQ ID NO: 146.

In some embodiments, the antigen binding region that specifically binds TROP2 comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are HCDR1, HCDR2, and HCDR3 of a heavy chain variable region as set forth in SEQ ID NO: 128.

In some aspects, the antigen binding region that specifically binds TROP2 comprises or consists of a heavy chain variable region (VH) , wherein the VH

(i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 128, or

(ii) comprises or consists of an amino acid sequence of SEQ ID NO: 128.

In some embodiments, the antigen binding region that specifically binds TROP2 is a VHH which comprises or consists of the amino acid sequence of SEQ ID NO: 128.

Illustrative multi-specific proteins

In some embodiments, the multi-specific protein in the present disclosure includes the following:

*also includes EM1030-NIH-42 to -58, containing different linkers, as illustrated in Table 13.

Properties of the multi-specific proteins

The multi-specific proteins of the present disclosure have one ore more of the following properties:

(1) reduced IL15 potency;

(2) preparation and/or purification friendly, e.g., having higher purity after one-step Protein A purification;

(3) higher purity and/or yield;

(4) higher selectivity in activating NK cells, optionally while minimal IL15 activity on T cells,

(5) high selectivity for NK cells regarding IL15 activity;

(6) inducing NK cell cytotoxicity towards tumor cells;

(7) high-selective cytokine receptor signaling on NK cells with not T cells;

(8) less cytokine release risk;

(9) resulting significant tumor growth inhibition; and/or

(10) better tolerance, e.g., less or no decrease in body weight compared to vehicle control.

Immunoconjugate and immune fusion

In some embodiments, the present disclosure provides an immunoconjugate, comprising any binding protein of the present disclosure and other substances. In one embodiment, the other substance is an active ingredient.

In some embodiments, such an additional ingredient includes, but is not limited to, a prophylactic and/or therapeutic agent, a detection agent, such as an anti-tumor drug, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an antibody of different specificity or functional fragment thereof, a small molecule drug, an immunosuppressant or a detectable label or reporter. In one embodiment, the additional prophylactic or therapeutic agents are known to be useful for, have been used, or are currently being used in the prevention, treatment, management, or amelioration of, a disorder or one or more symptoms thereof.

In some embodiments, the immunoconjugate is an Antibody-Drug-Compound (ADC) .

Pharmaceutical compositions

The present disclosure also provides pharmaceutical compositions comprising an anti-NKp46 antibody, or antigen-binding portion thereof, or a multi-specific binding protein of the present disclosure (i.e., the primary active ingredient) and a pharmaceutically acceptable carrier. In a specific embodiment, a composition comprises one or more antibodies or binding proteins of the present disclosure.

Pharmaceutical compositions of the present disclosure may further comprise at least one additional active ingredient. In some embodiments, such an additional ingredient includes, but is not limited to, a prophylactic and/or therapeutic agent, a detection agent, such as an anti-tumor drug, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an antibody of different specificity or functional fragment thereof, a small molecule drug, an immunosuppressant or a detectable label or reporter. In one embodiment, the pharmaceutical composition comprises one or more additional prophylactic or therapeutic agents, i.e., agents other than the antibodies or binding proteins of the present disclosure, for the treatment or alleviation of a disorder. In one embodiment, the additional prophylactic or therapeutic agents are known to be useful for, have been used, or are currently being used in the prevention, treatment, management, or amelioration of, a disorder or one or more symptoms thereof.

The pharmaceutical compositions comprising proteins of the present disclosure are for use in, but not limited to, diagnosing, detecting, or monitoring a disorder; treating, managing, or ameliorating a disorder or one or more symptoms thereof; and/or research. In some embodiments, the composition may further comprise pharmaceutical acceptable supplementary material, such as a carrier, diluent, or excipient. An excipient is generally any compound or combination of compounds that provides a desired feature to a composition other than that of the primary active ingredient (i.e., other than an antibody, functional portion thereof, or binding protein of the present disclosure) .

Nucleic acid, vector, and host cells

In a further aspect, this disclosure provides isolated nucleic acids encoding one or more amino acid sequences of an anti-NKp46 antibody of this disclosure or an antigen-binding fragment thereof; or an isolated nucleic acid encoding one or more amino acid sequences of a multi-specific protein of the present disclosure. Such nucleic acids may be inserted into a vector for carrying out various genetic analyses or for expressing, characterizing, or improving one or more properties of an antibody or binding protein described herein. A vector may comprise one or more nucleic acid molecules encoding one or more amino acid sequences of an antibody or binding protein described herein in which the one or more nucleic acid molecules is operably linked to appropriate transcriptional and/or translational sequences that permit expression of the antibody or binding protein in a particular host cell carrying the vector. Examples of vectors for cloning or expressing nucleic acids encoding amino acid sequences of binding proteins described herein include, but are not limited to, pcDNA (e.g., pcDNA3.1) , pTT, pTT3, pEFBOS, pBV, pJV, and pBJ, and derivatives thereof.

The present disclosure also provides a host cell expressing, or capable of expressing, a vector comprising a nucleic acid encoding one or more amino acid sequences of an antibody or binding protein described herein. Host cells useful in the present disclosure may be prokaryotic or eukaryotic. An exemplary prokaryotic host cell is Escherichia coli. Eukaryotic cells useful as host cells in the present disclosure include protist cells, animal cells, plant cells, and fungal cells. An exemplary fungal cell is a yeast cell, including Saccharomyces cerevisiae. An exemplary animal cell useful as a host cell according to the present disclosure includes, but is not limited to, a mammalian cell, an avian cell, and an insect cell. Exemplary mammalian cells include, but are not limited to, CHO cells, HEK cells, and COS cells.

In some embodiments, mammalian host cells for expressing the recombinant antibodies of the present disclosure are Chinese Hamster Ovary (CHO cells) (including dhfr^–CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216-4220 (1980) , used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982) ) , HEK293 cells, NS0 myeloma cells, COS cells, and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period sufficient to allow for expression of the antibody in the host cells, or further secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules or variable regions of heavy chain or light chain. It will be understood that variations on the above procedure are within the scope of the present disclosure. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of either the light chain and/or the heavy chain of an antibody of this disclosure. Recombinant DNA technology may also be used to remove some, or all, of the DNA encoding either or both light and heavy chains that is not necessary for binding to the antigens of interest. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present disclosure.

Methods for production

In another aspect, the present disclosure provides a method of producing an anti-NKp46 antibody or a functional fragment thereof comprising culturing a host cell comprising an expression vector encoding the antibody or functional fragment in culture medium under conditions sufficient to cause the host cell to express the antibody or fragment capable of binding NKp46.

In another aspect, the present disclosure provides a method of producing a multi-specific protein of the present disclosure, comprising culturing a host cell comprising an expression vector encoding the multi-specific protein of the present disclosure in culture medium under conditions sufficient to cause the host cell to express the multi-specific protein capable of binding NKp46, the IL15R and the target antigen. The proteins produced by the methods disclosed herein can be isolated and used in various compositions and methods described herein.

In an exemplary system for recombinant expression of the antibody or antigen-binding portion thereof or the multi-specific protein of the present disclosure, a mixture of the recombinant expression vectors encoding each of the chains of the antibody or the antibody-binding portion thereof or the multi-specific protein is introduced into HEK293 or CHO by transfection. The selected transfected host cells are cultured to allow expression of the chains of the antibody or the antibody-binding portion thereof or the multi-specific protein and intact antibody or antigen-binding portion thereof or multi-specific protein is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transfectants, culture the host cells and recover the antibody or the antibody-binding portion thereof or the multi-specific protein from the culture medium. The present disclosure further provides a method of making an antibody or antigen-binding portion thereof or a multi-specific protein by culturing a transfected host cell of the present disclosure in a suitable culture medium until a recombinant multi-specific protein of the present disclosure is produced. The method can further comprise isolating the recombinant antibody or antigen-binding portion thereof or multi-specific protein from the culture medium.

In an exemplary system for recombinant expression of an antibody, or antigen-binding portion thereof or a multi-specific protein, of the present disclosure, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain or each chains of the proteins is introduced into dhfr^–CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes or each chain genes of the proteins are each operatively linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transfected host cells are cultured to allow expression of the antibody heavy and light chains or the protein chains and intact antibody or multi-specific protein is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transfectants, culture the host cells and recover the antibody or the multi-specific protein from the culture medium. The present disclosure further provides a method of making a recombinant anti-NKp46 antibody or multi-specific protein by culturing a transfected host cell of the present disclosure in a suitable culture medium until a recombinant antibody of the present disclosure is produced. The method can further comprise isolating the recombinant antibody or the multi-specific protein from the culture medium.

Uses of antibodies and multi-specific proteins

Given their ability to bind to NKp46, the anti-NKp46 antibody or antigen binding fragments described herein, can be used to detect NKp46, e.g., in a biological sample containing cells that express NKp46.

The anti-NKp46 antibody or antigen binding fragments of the present disclosure can be used in a conventional immunoassay, such as an enzyme linked immunosorbent assay (ELISA) , a radioimmunoassay (RIA) , or tissue immunohistochemistry. The present disclosure provides a method for detecting NKp46 in a biological sample comprising contacting a biological sample with an anti-NKp46 antibody or antigen binding fragments of the present disclosure and detecting whether binding to a target antigen occurs, thereby detecting the presence or absence of the target in the biological sample.

Given their ability to bind to NKp46, IL15R and the other antigens, the multi-specific proteins described herein, can be used to detect NKp46, IL15R and/or the other antigens, e.g., in a biological sample containing cells that express one or both or all of those target antigens.

The multi-specific proteins of the present disclosure can be used in a conventional immunoassay, such as an enzyme linked immunosorbent assay (ELISA) , a radioimmunoassay (RIA) , or tissue immunohistochemistry. The present disclosure provides a method for detecting NKp46 and the other antigens in a biological sample comprising contacting a biological sample with a multi-specific protein of the present disclosure and detecting whether binding to a target antigen occurs, thereby detecting the presence or absence of the target (s) in the biological sample.

The anti-NKp46 antibody or the antigen binding fragments thereof, or multi-specific protein may be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody/fragment/multi-specific protein. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

In some embodiments, the present disclosure provides an anti-NKp46 antibody or antigen binding fragments or multi-specific protein of the present disclosure for use in activating NK cells. In some embodiments, the present disclosure provides an anti-NKp46 antibody or antigen binding fragments or multi-specific protein of the present disclosure for use in treating any subject that would benefit from the activation of NK cells.

The anti-NKp46 antibody or antigen binding fragments or multi-specific proteins of the present disclosure can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises an antibody or multi-specific protein of the present disclosure and a pharmaceutical acceptable supplementary material.

In one embodiment, the pharmaceutical acceptable supplementary material is a pharmaceutically acceptable carrier.

As used herein, “pharmaceutically acceptable carrier” includes any or all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be useful to include isotonic agents, for example, sugars, polyalcohol (such as, mannitol or sorbitol) , or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the antibody or multi-specific protein present in the composition. A pharmaceutical composition of the present disclosure is formulated to be compatible with its intended route of administration.

The method of the present disclosure may comprise administration of a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion) . Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the primary active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

The use of the present disclosure may include administration of compositions formulated as depot preparations. Such long-acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. For example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt) .

An antibody, functional fragment thereof, or multi-specific protein of the present disclosure also can be administered with one or more additional therapeutic agents useful in the treatment of various diseases. Antibodies, functional fragments thereof, and multi-specific proteins described herein can be used alone or in combination with one or more therapies, such as surgery, radiotherapy or an additional agent. The additional agent being selected by the skilled artisan for its intended purpose. For example, the additional agent can be a therapeutic agent recognized in the art as being useful to treat the disease or condition being treated by the antibody or multi-specific protein of the present disclosure. The additional agent also can be an agent that imparts a beneficial attribute to the therapeutic composition, e.g., an agent that affects the viscosity of the composition. In one embodiment, the additional agent includes, but is not limited to, a prophylactic and/or therapeutic agent, a detection agent, such as an anti-tumor drug, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an antibody of different specificity or functional fragment thereof, a small molecule drug, an immunosuppressant or a detectable label or reporter.

The present disclosure may thus provide a combination product or kit comprising

i. antibody, functional fragment thereof, or multi-specific protein of the present disclosure; and

ii. one or more additional ingredient useful in the treatment of various diseases, e.g., one or more additional therapeutic agents.

In some embodiments, such an additional ingredient includes, but is not limited to, a prophylactic and/or therapeutic agent, a detection agent, such as a prophylactic and/or therapeutic agent, a detection agent, such as an anti-tumor drug, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an antibody of different specificity or functional fragment thereof, a small molecule drug, an immunosuppressant or a detectable label or reporter. In one embodiment, the additional prophylactic or therapeutic agents are known to be useful for, have been used, or are currently being used in the prevention, treatment, management, or amelioration of, a disorder or one or more symptoms thereof.

Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting of the present disclosure.

Methods for treatment and medical uses

In some embodiments, the present disclosure provides methods for treating cancers or tumors in a subject in need thereof, the method comprising administering to the subject anti-NKp46 antibody or antigen binding fragments, multi-specific proteins of the present disclosure, disclosed herein, pharmaceutical composition or combination product. The cancer to be treated in accordance with the present disclosure may be any unwanted cell proliferation, neoplasm or tumor. The cancer may be benign or malignant and may be primary or secondary. The cancer may be metastatic.

In some embodiments, the cancer to be treated can be solid cancer or hematopoietic cancer. In some embodiments, the cancer to be treated is a solid cancer. In some embodiments, the cancer to be treated may be a cancer of a tissue selected from the group consisting of colon, rectum, cervix, oropharynx, nasopharynx, liver, stomach, head and neck, oral cavity, oesophagus, lip, mouth, tongue, tonsil, nose, throat, salivary gland, sinus, pharynx, larynx, prostate, lung, bladder, skin, kidney, ovary or mesothelium.

In some embodiments, the cancer to be treated include but not limited to, for example, melanoma, metastatic melanoma, renal cell carcinoma, ovarian cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, brain cancer, head and neck cancer, breast cancer (including three-negative breast cancer) , colon cancer, rectal cancer, colorectal cancer, skin cancer, throat cancer, uterine cancer, cervical carcinoma, hepatocellular carcinoma, prostate cancer, pancreatic cancer, gastric cancer, chronic lymphocytic leukemia (CLL) , multiple myeloma, lymphoma, or bladder cancer.

Methods of treatment described herein may further comprise administering to a subject in need thereof, of additional therapy, e.g., surgery, radiotherapies or additional active ingredient, which is suitably present in combination with the present antibody or multi-specific protein for the treatment purpose intended, for example, another drug having ant-tumor activity. In a method of treatment of the present disclosure, the additional active ingredient may be incorporated into a composition comprising an antibody or multi-specific protein of the present disclosure, and the composition administered to a subject in need of treatment. In another embodiment, the additional active ingredient can be comprised in a combination product or kit, in combination with an antibody or multi-specific protein of the present disclosure. In another embodiment, a method of treatment of the present disclosure may comprise a step of administering to a subject in need of treatment an antibody or multi-specific protein described herein and a separate step of administering the additional active ingredient to the subject before, concurrently, or after the step of administering to the subject an antibody or multi-specific protein of the present disclosure.

Examples

Example 1: Generation and characterization of anti-NKp46 antibody

1.1 Immunization and hybridoma generation

Anti-NKp46 antibodies were obtained by immunizing groups of Balb/c mice with the recombinant fusion protein of NKp46 isoform4 Extra Cellular Domain (ECD) and human IgG1 Fc (SEQ ID NO: 1) .

Mice were immunized at 2-week intervals and monitored for serum titer once a week after the second injection. After 4 to 6 immunizations, splenocytes were harvested and fused with mouse myeloma cells to form hybridoma cell lines. Supernatants of hybridoma cells were then screened against the CHO-k1 cells (ATCC, cat#CCL-61) transfected with human NKp46 and counter-selected with un-transfected CHO-k1 cells (ATCC, cat#CCL-61) and recombinant human IgG1 Fc to identify hybridomas that produce NKp46-specific antibodies.

Positive hybridomas were then tested for cynomolgus cross-reactivity by ELISA with a recombinant fusion protein of cynomolgus NKp46 ECD and human IgG1 Fc (SEQ ID NO: 2) as a target, and a Jurkat-NFAT-Luciferase reporter cell line (ChemPartner, Shanghai, China) was used to characterize anti-NKp46 agonistic activity.

Briefly, 96-well plates were pre-coated with serial diluted antibodies at 4℃ overnight. The plates were washed twice by PBS. Then Jurkat-NFAT-Luciferase reporter cells were added into the plates at 4×10⁴ cells per well and incubated at 37℃ for 4 hours. ONE-GloTM luminescence assay kit (Promega, Cat#E6130) reagents were prepared according to the manufacturer’s instructions and added into wells. Luminescence signals were measured with a microplate reader.

Upon preliminary characterization of the antigen-specific binding to NKp46, one hybridomas clone named NKp46-mab03 NKp46was producing monoclonal antibodies that tested positive in all assays, which were selected and sequenced.

1.2 Chimeric anti-NKp46 and reference antibody generation

VH and VK genes of NKp46-mab03 were respectively synthesized and cloned into vectors containing human IgG1 heavy chain constant domain with LALA (L234A, L235A) mutation (SEQ ID NO: 3) and human kappa constant domain (SEQ ID NO: 4) . 293E cells co-transfected with both heavy chain vector and light chain vector were cultured 6 days, then the supernatant was harvested and purified by Protein A chromatography. The purified chimeric antibodies were designated EM1010-mab03c respectively.

Reference anti-NKp46 antibody named WT3 containing the variable regions of NKp46-3 (as described in International PCT Publication NO. WO2017114694) was constructed following the above procedure except that WT3 contains human IgG1 heavy chain (wild type) and human kappa constant domains.

Amino acid sequences of EM1010-mab03c and WT3 variable regions were listed in Table 1. Complement determinant regions (CDRs) were underlined according to Kabat scheme.

Table 1: Amino acid sequences of variable regions of anti-NKp46 antibodies

*CDRs are underlined (determined according to Kabat scheme) .

1.3 Characterization of chimeric and reference anti-NKp46 antibodies

1.3.1 Binding off-rate measurement of anti-NKp46 antibodies to both human and cynomolgus NKp46

Binding kinetics constants of anti-NKp46 antibodies were determined at 25℃ using anbiolayer interferometry (Pall FortéBio LLC) following standard procedures. Briefly, Anti-Mouse IgG Fc Capture (AMC) Biosensors were used to capture purified anti-NKp46 antibodies. Sensors were then dipped into solutions containing recombinant human NKp46-ECD protein to detect target protein binding to the captured antibodies. Kinetics constants were determined by processing and fitting data to a 1: 1 binding model using Fortebio analysis software.

Table 2: Binding off-rate of anti-NKp46 antibodies against both human and cynomolgus NKp46

1.3.2 Cell surface binding characterization of anti-NKp46 antibodies

Cell binding activity of anti-NKp46 antibodies were measured with a human NKp46 transfected CHO-k1 cell line (CHOk1-NCR1-3E11) and a cynoNKp46 transfected CHO-k1 cell line (CHOk1-EGFP-cynoNKp46-B9) . Briefly, 5×10⁵ cells in F12-K with 10%FBS (Gibco, cat#10099-141C) were seeded into each well of a 96-well plate. Cells were centrifuged at 400g for 5 minutes and supernatants were discarded. 100 μL of 3x serially diluted antibodies (diluted from 100nM, in 7 antibody concentrations) were then added and mixed with the cells. After 40 minutes of incubation at 4℃, plates were washed several times to remove excessive antibodies. Secondary fluorochrome-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch, Cat#109-606-098) was then added and incubated with cells at room temperature for 20 minutes. After another round of centrifugation and washing step, cells were resuspended in FACS buffer (1x PBS containing 2%FBS) and measured with a CytoFLEX Flow Cytometer (Beckman Coulter) . Median Fluorescence Intensity (MFI) readouts were plotted against antibody concentration and analyzed with GraphPad Prism 8.0 software. Binding EC50 were listed in Table 3.

Table 3: Cell binding EC50 of anti-NKp46 antibodies

1.3.3 Epitope binning of anti-NKp46 antibodies

Epitope binning of anti-NKp46 mAbs (including EM1010-mab03c, WT3, and 9E2) were characterized with a competition ELISA. 9E2 mIgG1 (Short for “9E2” hereinafter) is available commercially from Biolegend (California, US, cat#331902) .

Briefly, 96-well plates were coated with 1μg/mL purified antibodies and incubated overnight at 4℃. After washing with PBS containing 0.05%Tween 20, plates were blocked with blocking buffer (PBS containing 0.05%Tween 20 and 1%BSA) at room temperature for 1 hours. Biotinylated human NKp46-ECD protein pre-mixed with anti-NKp46 antibodies (sample) or irrelevant human IgG1 (baseline) was added and incubated at room temperature for 1 hour. After 3 times plate wash, Streptavidin-HRP (Sigma, S2438) (1: 5000 dilution) was added into each well and incubated at room temperature for 30 minutes and followed by another 3 times plate wash. Tetramethylbenzidine (TMB) chromogenic solution (Biopanda, TMB-S-003) was added for color development for 2 minutes then the reaction was stopped with 1M HCl. Absorbance at 450 nm (OD₄₅₀) was measured on a microplate reader. The OD450_baseline represents the level of human NKp46-ECD binding to anti-NKp46 antibodies at absence of competition, while the difference between OD450_baseline and OD450_sample reflects the competition between the NKp46 antibody coated on plate and the antibody in solution. The inhibition percentage was calculated by following equation and shown in Table 4:

Inhibition %= (1 -OD450_sample /OD450_baseline) × 100%

Table 4: Epitope binning of anti-NKp46 antibodies

Table 4 shows results of the competition ELISA in terms of percent inhibition, indicating that EM1010-mab03c compete with WT3, but do not compete with 9E2.

Example 2: Humanization and modification of anti-NKp46 antibody

2.1 Humanization of EM1010-mab03c

The EM1010-mAb03c variable region genes provided in Table 1 were employed to create a humanized antibody. First, amino acid sequences of the VH domain and the VK (VL kappa) domain of EM1010-mAb03c were compared against available human Ig V-gene sequences from V BASE database (https: //www2. mrc-lmb. cam. ac. uk/vbase/alignments2. php) in order to find the overall best-matching human germline Ig V-gene sequences. The framework segments of VH and VK were also compared against available FR sequences in the J-region sequences in V BASE to find the human framework having the highest homology to the murine VH and VK regions, respectively. For the light chain, the closest human V-gene match was the A10 gene; and for the heavy chain, the closest human match was the VH1-8 gene. Humanized variable domain sequences were then designed to have the CDR-L1, CDR-L2, and CDR-L3 of the EM1010-mab03c light chain grafted onto framework sequences of the A10 gene and JK2 framework 4 sequence while the CDR-H1, CDR-H2, and CDR-H3 of the EM1010-mAb03c heavy chain grafted onto framework sequences of the VH1-8 and JH6 framework 4 sequence.

Meanwhile, a three-dimensional Fv model of EM1010-mAb03c was generated to identify any framework positions where mouse amino acids were critical to support loop structures or the VH/VK interface. Corresponding residues within the human framework sequences should be back-mutated to the mouse residues at such identified positions to retain affinity/activity. Several desirable back-mutations were indicated for EM1010-mAb03c VH and VK, and alternative VH and VK designs were constructed as shown in Table 5 below. Since the "NG" (Asn-Gly) pattern found in the CDR-H2 of EM1010-mAb03c is prone to deamination and may result in heterogeneity during manufacturing, VH and VL domains containing an NG (Asn-Gly) to NA (Asn-Ala) mutation (highlighted in bold italic) , e.g., designated "EM1010-mAb03VH (G-A) ” , were also designed and evaluated.

Table 5: Humanized VH/VK designs for EM1010-03 with back mutations*

*CDR sequences according to the Kabat numbering system single underlined; framework back-mutationsNG (Asn-Gly) to NA (Asn-Ala) mutation in bold italic.

The humanized VH and VK (VL kappa) genes were synthesized and then respectively cloned into vectors containing the human IgG1 heavy chain constant domains with LALA mutation (L234A, L235A) (SEQ ID NO: 3) and the human kappa light chain constant domain (SEQ ID NO: 4) .

Pairing of the humanized VH and the humanized VK chains created 16 humanized antibodies, designated "HuEM1010-03-1" to "HuEM1010-03-16" as shown in Table 6 below.

Table 6: Anti-NKp46 chimeric and humanized EM1010-03 antibodies

All 16 antibodies in Table 6 were expressed by transient transfection of HEK293 cell, purified by Protein A chromatography, and assayed for and ranked by dissociation rate constant (k_off) . Briefly, antibodies binding affinities and kinetics were characterized by biolayer interferometry (Pall FortéBio LLC) . Antibodies were captured by Anti-hIgG Fc Capture (AHC) Biosensors (Pall) at a concentration of 100 nM for 120 seconds. After that, sensors were dipped into running buffer (1X pH 7.2 PBS, 0.05%Tween 20, 0.1%BSA) for 60 seconds to check the baseline, then dipped into recombinant human NKp46/His fusion protein (Novoprotein, CB17) at assigned concentration for 200 seconds to measure binding, followed by dipped into running buffer for 600 seconds for dissociation. The assay was conducted containing the EM1010-mab03c chimeric antibody as the basis for normalization. The association and dissociation curves were fitted to a 1: 1 Langmuir binding model using FortéBio Data Analysis software (Pall) to obtain the off-rate constants as shown in Table 7 below. The off-rate of each antibody was compared to that of the EM1010-mAb03c chimeric antibody in the same test group obtained in parallel to produce the corresponding off-rate ratio, serving as a normalized index. The normalized index of an antibody indicates higher affinity for human NKp46.

Table 7: Off-rates (koff) of humanized and chimeric EM1010-03 antibodies

2.2 Characterization of humanized anti-NKp46 antibodies

2.2.1 Binding off-rate measurement of humanized anti-NKp46 antibodies to both human and cynomolgus NKp46

Binding kinetics constants of humanized anti-NKp46 antibodies were determined following standard procedures as described in Example 1.3.1. Table 8 below illustrates binding off-rates of humanized anti-NKp46 antibodies.

Table 8: Binding off-rate against both human and cynomolgus NKp46

2.2.2 Cell surface binding characterization of humanized anti-NKp46 antibodies

Cell binding activity of humanized anti-NKp46 antibodies were measured with a human NKp46 transfected CHO-k1 cell line (CHOk1-NCR1-3E11) and a cynoNKp46 transfected CHO-k1 cell line (CHOk1-EGFP-cynoNKp46-B9) . Cell staining and measurement followed the procedure as described in Example 1.3.2, and cell binding EC50s were shown in Table 9.

Table 9: Cell binding EC50 of anti-NKp46 antibodies

2.2.3 Humanized anti-NKp46 antibodies cross-reactivity

Cross-reactivity of anti-NKp46 mAbs (including EM1010-mab03c, HuEM1010-03-9, HuEM1010-03-11, and ref WT3) were characterized with an ELISA assay, irrelevant hIgG1 was used as negative control.

Briefly, 96-well plates were coated with 2μg/mL humanNKp46-his, cynoNKp46-his, mouseNKp46-his, respectively, and incubated overnight at 4℃, overnight. After washing with PBS containing 0.05%Tween 20, plates were blocked with blocking buffer (PBS containing 0.05%Tween 20 and 1%BSA) at room temperature for 1 hour. Anti-NKp46 mAbs or irrelevant human IgG1 were added and incubated at room temperature for 1 hour. After 3 times plate wash, anti-humanFc-HRP (1: 5000 dilution, Sigma, Cat#A0170-1ML) was added into each well and incubated at room temperature for 30 minutes and followed by another 3 times plate wash. Tetramethylbenzidine (TMB) chromogenic solution was added for color development for 2 minutes then the reaction was stopped with 1M HCl. Absorbance at 450 nm (OD₄₅₀) was measured on a microplate reader. The OD₄₅₀ reflects the anti-NKp46 mAbs (EM1010-mab03c, HuEM1010-03-9, HuEM1010-03-11) showed positive binding activity to humanNKp46-his, cynoNKp46-his, but negative binding activity to mouseNKp46-his. The absorbance data were shown in Table 10.

Table 10: Cross-reactivity of humanized anti-NKp46 antibodies

2.3 Chimeric and humanized anti-NKp46 antibodies activated NK cells

Chimeric NKp46 antibodies having a mouse anti-human NKp46 Fab2 domain and a human IgG1 Fc domain (L234A, L235A) (EM1010-mab03c) and humanized anti-human NKp46 antibodies having a humanized anti-human NKp46 Fab2 domain and a human IgG1 Fc domain (L234A, L235A) (HuEM1010-03-9) were further tested for functional activity to stimulate NK cell cytotoxic degranulation by measuring membrane CD107a expression.

Briefly, the functional activity of resting human NK cells was assessed in a CD107a flow cytometry assay in high-binding 96 well plates. Chimeric NKp46 antibodies (EM1010-mab03c) , humanized NKp46 antibodies (HuEM1010-03-9) and isotype control antibody (Humanized hIgG1 with no-NKp46 binding) not binding to NK cells were diluted in DPBS to 3μg/ml and 1μg/ml and then 100ul of each diluted antibody solutions were added to high-binding 96 well plates (Corning 3361) . After overnight incubation at 4℃, the plates were washed twice with DPBS. Resting NK cells isolated from healthy donor PBMC (MT bio PB100C) with CD56 microbeads (Miltenyi, 130-050-401) and then resuspended in X-Vivo 15 media (Lonza, 04-418Q) at 1.5*10⁶ cells/ml. One hundred microliter of the cell suspension was added to the antibody bound plates. After 4 hours of incubation at 37℃, NK cells were washed once with FACS buffer (DPBS plus 2%FBS) and resuspended with 100μl of FACS buffer containing 2.5μL of Human TruStain FcX (Biolegend, 422302) . After 5 minutes of incubation, 2.5μl CD107a detection antibody (Biolegend, 328620 and 328626) or isotype control were added to the cells. After 20 minutes of incubation at 4℃, the cells were washed FACS buffer and applied to flow cytometry (Beckman, Cytoflex) for detection. Percentages of CD107a positive NK cells were calculated for each treatment.

Plate-bound chimeric NKp46 antibodies (EM1010-mab03c, see Figure 1A) , humanized NKp46 antibodies (HuEM1010-03-9, see Figure 1B) up-regulated membrane CD107a expression on NK cells, which is a marker of cytotoxic degranulation and immune cell activation, indicating NK cells could be activated by the chimeric and humanized NKp46 antibodies.

Example 3: IL15 variants (vs. wild-type)

An IL15 variant (IL15v) , along with a sushi domain, was constructed as a heterodimeric Fc-domain-containing protein comprising an IL15v fused to N-terminus of one Fc chain, and an IL-15Rα sushi domain fused to N-terminus of the other Fc chain, as shown in Figure 2. To reduce the effector function of Fc and form a heterodimeric protein, amino acid substitutions L234A, L235A, L351D and L368E were introduced to one Fc chain and amino acid substitutions L234A, L235A, L351K and T366K were introduced to the other Fc chain. A particular IL15-containing protein IL15-N5 with a wild-type IL15, as a control, was constructed as described above (Figure 2) .

Various IL15 variants based on human IL-15 and modified by introducing mutation D61A, E64A, N65A, I68A, L69A, N72A, or N65A + L69A, were incorporated in the protein as described in Figure 2, and designated as IL15-N21, IL15-N22, IL15-N23, IL15-N24, IL15-N25, IL15-N26 and IL15-N39, respectively.

To test IL15 potency of these IL15vs, an IL15 reporter gene assay was performed. Briefly, 50 thousand of HEK-Blue^TM IL-2 Cells (Invivogen, #hkb-il2) were seeded into a 96-well plate and followed by adding serially diluted IL15v protein. After overnight incubation in a tissue culture incubator, 25μl of supernatant was transferred to a new 96-well plate with flat and clear bottom containing 180μl of QUANTI-Blue solution (InvivoGe #rep-qbs) . The plate was incubated at 37℃ for 30 minutes and read for OD630 signal with a plate reader (Molecular Devices, #33 270 -3091) . The absorbance at 630nM were plotted against test articles concentration with GraphPad Prism software. The exemplary dose-response curves were shown in Figure 3 and the calculated EC₅₀ values were listed in Table 11. The results indicate that IL15vs with single site mutation such as N65A or L69A are potency-reduced variants with 6.1-fold and 4.5-fold decreases respectively, in potency compared to wild-type IL15 (IL15-N5) .

Table 11: EC₅₀ summary of IL-15v potency determination

Example 4: Design and generation of NKCE

4.1 Various formats of multi-specific proteins

To obtain multi-specific proteins capable of selectively targeting NK cells and increasing NK cell cytotoxicity toward a target cell, various formats of multi-specific proteins were designed to bind NKp46, IL15 receptor on NK cells, and an antigen of interest (e.g., a cancer/tumor antigen) on a target cell (e.g., a tumor/cancer cell) , optionally further bind CD16A at the surface of NK cells.

Ideally, the multi-specific proteins comprise a potency-reduced IL15 variant (IL15v) and antigen-binding domains (ABD) , which can bind, in cis, to the cytokine receptor and NKp46, optionally to CD16A, conferring a restored IL15 potency, allowing said protein high-selective cytokine receptor signaling on NK cells while not T cells.

In light of various combined configurations of IL15v, NKp46 binding domain, target antigen binding domain, and Fc domain (IgG1) , five major Fc-domain-containing formats (Formats I-IV and T5) , including several sub-formats were designed and shown in Figures 4-9.

In Format I of multi-specific protein with exemplary I-1, I-2, and I-3, IL-15 is placed on N-terminus of Fc domain containing Fc-engineered mutations favoring heterodimerization (e.g., DEKK, DDKK, and/or KIH mutations) , and optionally those to render an Fc effector function silence (e.g., LALA and/or P329A/G mutations) .

Multi-specific protein of Format I-1 contains, NKp46 and target antigen-binding Fabs in-tandem fused to N-terminus of Fc domain, while IL-15 linking a sushi domain is fused to N-terminus of the other Fc chain. A Format I-2 protein comprises an IL15-sushi domain complex separately positioned to N-terminus of each Fc chain; one of NKp46 and target antigen binding domains (Fab) is linked to IL-15, and the other to sushi domain at N-terminus of IL15/sushi domain, respectively. A Format I-3 protein comprises a sushi domain located at N-terminus of one Fc chain complexed with IL-15 harboring an interchain-di-sulfide bridge formed by amino acid substitutions of IL15 (L52C) and sushi domain (S40C) polypeptide chains, as well as a human NKp46 or target antigen domain (Fab) positioned at N-terminus of the other Fc chain. In addition, VH/VL regions (or VL/VH) of target antigen or NKp46 binding domain are linked to IL-15 and sushi domain at N-terminus thereof, respectively (see Figure 4) .

Similarly, Format II multi-specific protein has its IL15 and/or sushi domain (if exists) located separately (optionally by a glycine-serine linker such as G4S, (G4S) ₂) at C-terminus of each Fc (effector function silenced) domain, forming IL15-sushi complex by non-covalent binding. NKp46 binding domain (Fab or scFv) and target antigen binding domain (Fab, or scFv) are fused separately to the N-terminus of two Fc chains. Sub-Formats II-1 to II-4 vary from each other by IL-15 mutations, linkage of IL-15, as shown in Figure 5.

Format III-1 is similarly designed as Format II-2 except for having effector function-competent Fc domain, and in parallel, the scFv domain of Format III-1 is substituted by a VHH, leading to Format III-2. Meanwhile, Format III-1a and III-2a without IL15-sushi complex serve as no-IL15 moiety controls of III-1 and III-2, while Formats III-1b, III-1c, and III-1d are designed respectively as no-NKp46-binding, no-sushi-domain, and Fc-enhanced reference molecules in comparison with III-1 (see Figure 6) .

Multi-specific proteins of Format IV comprise IL15 and sushi domain linked to one another by a linker from N-terminus to C-terminus, forming a complex. The IL15-sushi complex is fused to C-terminus of one Fc chain of an IgG1 mab containing two Fabs of NKp46/target antigen binding domain. A target antigen/NKp46-binding scFv is linked to C-terminus of the other Fc chain. Optionally, the Fc is function silenced (e.g., with LALA, and/or P329A/G mutations) . Exemplary constructions of Format IV are shown in Figure 8, where IV-1, IV-2, and IV-3 vary by IL-15 mutations.

Format T5 multi-specific proteins comprise, from N-terminus to C-terminus, antigen A-binding domain (Fab) , Fc domain capable of binding CD16a, antigen B-binding domain (VH-Cκ/Vκ-CH1 complex) and cytokine moiety, wherein T5-I to T5-III contain IL15v-sushi complex, IL15v (N65A) and IL2v (R38A+F42K+C125A) respectively; wherein one of antigens A and B is NKp46 and the other is a target antigen different from NKp46. IL15v/IL2v or sushi (if exists) is fused to C-terminus of polypeptide chain of Vκ-CH1 or VH-Cκ by a linker (see Figure 9) .

4.1.1 Format of multi-specific protein impacts IL-15 potency in reporter gene assay

Multi-specific proteins were constructed comprising target antigen-binding domain, Fc domain, NKp46-binding domain and IL15v (or wild type) .

IL15-N1 is a Format I-1 protein comprising an IL15-sushi domain complex fused one another via a (G4S) ₂ linker and placed on N-terminus of one Fc chain, and in-tandem Fabs targeting human NKp46 and human Her2 from N-terminus to C-terminus fused to N-terminus of the other Fc chain.

IL15-N2 is a Format I-2 protein comprising IL15-sushi domain complex, a human NKp46 binding domain (Fab) and a human Her2 binding domain (Fab) . Each of adjacent domains is fused to one another via a (G4S) ₂ linker.

IL-15-N4-1 is a Format I-3 multi-specific protein comprising IL15-sushi domain complex linked to N-terminus of one Fc chain by a (G4S) ₂ linker. The VH and VL domain form one human Her2 binding element and are linked to sushi domain and IL15 respectively by a (G4S) ₂ linker. A human NKp46 binding domain (Fab) was fused to N-terminus of the other Fc chain.

IL-15-N4-2 of Format I-3 shows high similarity with IL15-N4-1 except that NKp46 binding domain and Her2 binding domain are swapped, wherein the VH and VL domains forming human NKp46-binding element are linked respectively to sushi domain and IL15 by a (G4S) ₂ linker.

EM1030-NIH-N2, -N3, -N4, -N8 and -N11 are Format II-1, II-2, II-3, II-4, and III-1 proteins, and EM1030-NIH-N5, -N9 and -N10 are Format IV-1, IV-2 and IV-3 proteins respectively, targeting human Her2.

The Her2-binding domain used in these molecules above is derived from trastuzumab.

All the molecules of T5-5 in Format III-1, T5-7/T5-8 proteins in Format T5-I, T5-9/T5-10 in Format T5-II, and T5-2 (IPH6501 analog) in Format T5-III target CD20. The NKp46 binding domain of T5-2, T5-5, T5-7, and T5-9 is derived from NKp46-1 mab described in International PCT Publication NO. WO2022258673A1, while HuEM1010-03-9 is used in T5-8 and T5-10. The CD20-binding domain in T5-2, T5-5, T5-7/8, and T5-9/T5-10 is derived from CD20-2 mab mentioned in WO2022258673A1.

Detailed information and amino acid sequences of all the molecules described above are summarized in Table 14 and Table 15.

To test IL15 potency of multi-specific proteins from Format I to IV, an IL15 reporter gene assay was performed as described in Example 3. The calculated EC₅₀ values were shown in Figure 10. Format II, III and IV multi-specific proteins showed a reduced IL15 potency compared with IL15-N5 control (wild-type) .

Reduced IL15 potency of EM1030-NIH-N2 in comparison with IL15-N5 indicates a steric hindrance effect on wild-type IL15 when positioned at N-terminus of Fc domain. By combining with IL15 mutation (N65A or L69A) , the IL15 potency could be further reduced (see EM1030-NIH-N3 and -N11 in Figure 10) . The data shows that the length of the linker between Fc domain and IL15v also impacts IL15 potency, indicating a shorter linker or even no linker for less potency (EM1030-NIH-N3 and -N4 in Figure 10) . IL15 potency in Format IV is reduced as well, whereas Format IV proteins give low purity after one-step Protein A purification compared with proteins in Format II/III (EM1030-NIH-N2 (Format II-1) , 75.69%vs. EM1030-NIH-N10 (Format IV-3, 29.64%) .

4.1.2 Format of multi-specific protein impacts IL-15 potency in T and NK cells

The multi-specific proteins mentioned in Example 4.1.1 were assessed for its ability to activate NK cells, CD4+ T cells and CD8+ T cells.

Briefly, 0.6 ×10⁶ cell/well of purified PBMC were seeded in 96-well plate and treated with increasing doses of multi-specific proteins for 15min at 37℃, 5%CO₂ in incubator. STAT5 phosphorylation was then analysed by flow cytometry on NK cells (CD3-CD56+) , CD8 T cells (CD3+ CD8+) and CD4 T cells (CD3+ CD4+) using BD Phosflow^TM Alexa 647 Mouse Anti-Stat5 (pY694) (cat#562076) .

The MFI (Mediate Florence Intensity) of pSTAT5 was plotted against the concentration of multi-specific proteins and the calculated EC50 was listed in Table 12. The data indicates that EM1030-NIH-N11 protein shows higher selectivity than other formats in activating NK cells, while minimal IL15 activity on T cells.

Table 12:

4.2 Fc variation

Multi-specific proteins targeting Her2 were designed with IL15v mutation (N65A) , plus a (G4S) ₂ linker between Fc domain and IL15/sushi moiety; and said molecules were constructed with various Fc mutations, such as EM1030-NIH-N57 in Format III-1d with IgG1 Fc carrying DLE mutations (a. a. substitutions, effector function enhanced) , EM1030-NIH-N3 in Format II-2 with IgG1 Fc carrying LALA mutations (effector function silenced) and EM1030-NIH-N11 in Format III-1 with IgG1 (normal effector function) .

Briefly, 0.6×10⁶ cells/well of purified PBMC were seeded in 96-well plate and treated with increasing doses of multi-specific proteins for 15min at 37℃, 5%CO₂ in incubator. STAT5 phosphorylation was then analysed by flow cytometry on NK cells (CD3-CD56+) , CD8 T cells (CD3+ CD8+) and CD4 T cells (CD3+ CD4+) using BD Phosflow^TM Alexa 647 Mouse Anti-Stat5 (pY694) (cat#562076) .

The MFI (Mediate Florence Intensity) of pSTAT5 was plotted against the concentration of multi-specific proteins.

4.3 Multi-specific proteins targeting various TAAs

4.3.1 Various linkers

To identify how linkers between Fc domain IL15v/sushi impact IL15v activity, multi-specific proteins in Format III-1 with null, G4S, (G4S) ₂ or (G4S) ₃ linker were designed, as shown in Figure 7.

In particular, proteins EM1030-NIH-N42 to -N56 were constructed based on EM1030-NIH-11, except for the linkers between Fc domain and IL15v/sushi domain (see Table 13 for detail information) .

Briefly, 50 thousand of HEK-Blue^TM IL-2 Cells (Invivogen, #hkb-il2) were seeded into a 96-well plate and followed by adding serially diluted IL15v protein (1000, 100, 10, 1, 0.1, 0.01, 0.001 and 0.0001nM) . After overnight incubation in a tissue culture incubator, 20ul of supernatant was transferred to a new 96-well plate with flat and clear bottom containing 180ul of QUANTI-Blue solution (InvivoGe #rep-qbs) . The plate was incubated at 37℃ for 30 minutes and read for OD₆₃₀ signal with a plate reader (Molecular Devices, #33 270 -3091) . The absorbance at 630nM was plotted against test articles concentration with GraphPad Prism software. The representative dose-response curves were shown in Figure 11 and the calculated EC₅₀ values were listed in Table 13 below. The data shows that IL15v activities are negatively correlated with the length of linkers. IL15-N5 containing a wild-type IL15 was used as a positive control.

Table 13: Production feasibility and IL-15 potency of proteins employing various linkers

4.3.2 Various target antigens

Multi-specific proteins with target antigen-binding domains of Her2, CD228, CD20, LIV-1, ALPP, CD123, and Trop2 were designed and constructed in Format III-1 or III-2 with IL15v mutation (N65A) , (G4S) ₂ linker between Fc domain and IL15/sushi domain, and IgG1 Fc with KIH mutation. The NKp46 binding domain (scFv) is derived from HuEM1010-03-09 (03-9) mentioned in Example 2.1.

EM1030-NIH-N11 contains a Her2 binding domain (Fab) derived from trastuzumab and an scFv NKp46 domain employing clone HuEM1010-03-9.

EM1030-NIC-N3 in Format III-1 is for CD228 targeting, where the CD228 binding domain (Fab) is derived from CD228 mab clone#SC57.10 mentioned in US 10, 428, 156 B2 and an scFv NKp46 domain employing clone HuEM1010-03-9. EM1030-NIC-N4 in Format III-1a is a no-IL15/sushi reference.

The LIV-1 binding domain (Fab) of EM1030-NIL-N3 in Format III-1 targeting LIV-1 is derived from ladiratuzumab and the scFv NKp46 domain is derived from HuEM1010-03-9. EM1030-NIL-N4 in Format III-1a is a no-IL15/sushi moiety control.

T5-5 targeting CD20 is a Format III-1 protein containing a CD20-binding domain (Fab) and an NKp46-binding scFv both derived from International PCT Publication NO. WO2022258673A1 with IL15v mutation (N65A) , (G4S) ₂ linker between Fc domain and IL15/sushi domain, and IgG1 Fc with KIH mutations.

EM1030-NIA-N3 of Format III-1 is for ALPP targeting, the sequence of ALPP-binding domain (Fab) is derived from ALPP mab (Clone #10ah-4) , and the scFv NKp46 domain is derived from HuEM1010-03-9. EM1030-NIA-N4 in Format III-1a is a no-IL15/sushi-moiety control.

EM1030-NIC123-N1 of Format III-1 is for CD123 targeting, the sequence of CD123-binding domain (Fab) is derived from talacotuzumab, and the scFv NKp46 domain is derived from HuEM1010-03-9.

EM1030-NIT-N30 is a Format III-2 protein targeting Trop2 containing a Trop2 VHH (VHH 2-5) and an NKp46 Fab using HuEM1010-03-9. EM1030-NIT-N31 in Format III-2a is a no-IL15/sushi-moiety control.

The results of reporter gene assay, and anti-tumor activity for the above multi-specific proteins are illustrated in Examples 6.1 and 6.3.

Exampe 5: Expression and purification of multi-specific proteins

5.1 Preparation of multi-specific proteins

A collection of multi-specific proteins was constructed and expressed in mammalian expression system. Molecule list and amino acid sequences are summarized in Tables 13, 14 and 15.

The sequences encoding each polypeptide chain of multi-specific protein were systhesized and cloned into the pcDNA3.1 mammalian expression vectors. The three or four recombinant expression vectors were cotransfected into HEK 293E cells. After approximately six days of post-transfection cell culture, the multi-specific proteins were purified from the supernatant following harvesting using Protein A (GE Healthcare, #17-5438-01) . Size Exclusion Chromatography (SEC) purifications were then performed, and the proteins eluted at the expected size were finally filtered on a 0.22 pm device.

5.2 Production feasibility of selected format

To test the production feasibility of selected format of multi-specific proteins, T5-5 in Format III-1, T5-7 and T5-8 in Format T5-I, T5-9 and T5-10 in Format T5-II were designed and generated as described in Example 4.1 and 5.1. After cell transfection, the multi-specific proteins were purified from the supernatant using one-step Protein A (GE Healthcare, #17-5438-01) . The purity of these proteins was detected by Size Exclusion Chromatography (SEC) as shown in Table 16. T5-5 in Format III-1 shows the highest purity and yield compared with Format T5-I/II.

Table 16: Comparison of production feasibility of selected formats

Example 6: Assessment and characterization of NKCE

6.1 Reporter gene assay

To test the IL15 receptor activation ability of above molecules, a reporter gene assay was performed as described in Example 3. The absorbance at 630nM was plotted against the concentration of protein and response curves and calculated EC₅₀ were shown in Figure 12A-12C. The data indicates EM1030-NIH-N11, -NIA-N3 and -NIC-N3 have the capability of activating IL15 signal pathway in dose-dependent manner and the potency is significantly lower than wild-type IL15 (IL15-N5) . EC₅₀ values of EM1030-NIH-N11, -NIA-N3 and -NIC-N3 were 3.616, 948.4 and 2.429nM respectively. Considering the expression of Her2 and CD228 in the reporter cells but not for ALPP (data not shown) , IL15 potency of EM1030-NIH-N11 and -NIC-N3 was enhanced, while EM1030-NIA-N3 was not.

6.2 Potency on T cells and NK cells

To assess the ability of activating NK and T cells, a STAT5 phosphorylation assay was performed for EM1030-NIH-N11 targeting Her2 as described in Example 4.1.2. EM1030-NIH-N34, -N3, -N16 and IL15-N5 were used as control articles. EM1030-NIH-N34 in Format III-1b with the scFv domain using a no-NKp46-binding domain is used as a negative control, EM1010-NIH-N3 in Format II-2 is an Fc-silenced control with LALA mutation in Fc domain, and -N16 in Format III-1a is a no-IL15/sushi moiety control.

The data indicates EM1030-NIH-N11 shows a reduced IL15 potency compared to IL15-N5 in NK cells and marginal IL15 activity for both CD8+ and CD4+ T cells, as shown in Figures 13A, 13B, and 13C. By binding to NKp46 and CD16a in a cis-configuration on NK cells, -N11 has enhanced IL-15 activity compared with -N34 (no NKp46 binding) and -N3 (Fc effector function silenced) . As a result, EM1030-NIH-N11 showed high selectivity for NK cells regarding IL15 activity.

6.3 Anti-tumor activities

6.3.1 Anti-tumor activities (in vitro)

EM1030-NIH-N11, -NIT-N30, -NIA-N3, -NIC-N3, and -NIC123-N1 molecules in Format III-1 or III-2, and EM1030-NIH-N16, -NIT-N31, -NIA-N4, -NIC-N4 controls were tested for anti-tumor activities. DF1001 analog (derived from A49-F3’ -TriNKET in WO2021041878, sequences listed in Table 15) , a tri-specific NK cell engager, containing Her2 binding domain (scFv derived from trastuzumab) , NKG2D binding domain (Fab) and effector function competent Fc, was used as a control.

Briefly, an NK-cell and tumor-cell co-culture assay were performed. The cytotoxic activity of human NK cells was assessed withLuminescent Cell Viability Assay (CTG) (Promega, Cat#G7572) in 96 well plates. Tumor cells (JIMT-1 (Cobioer, cat#CBP60378) , H1650, MDA-MB-231 (ATCC, HTB-26) , and Daudi) were mixed with human NK cells in culture medium (RPMI1640 + 10%FBS) . Serial diluted test articles were added into wells and incubated at 37℃ for 72 hours. After incubation, plate attached tumor cells were washed once with DPBS and cells viability was assessed with CTG assay kit. The luminescence was measured with a plate reader (Molecular Devices, Paradigm) .

To compare the capabilities of inducing NK cytotoxicity, IPH6501 analog (Format T5-III, also known as T5-2, see Tables 14 and 15) , a multi-specific protein containing a CD20-binding domain (derived from CD20-2 described in WO2022258673A1) , an NKp46-binding domain (Fab, derived from NKp46-1 in WO2022258673A1) , a functional IgG1 Fc, and an IL2 variant in tandem (IL2v2A in WO2022258673A1) , was used as a control. T5-5 and IPH6501 analog with serial concentrations (100, 12.5, 1.56, 0.195, 0.0244, 0.00305, 0.000381, and 0.0000477nM) were added to human NK and Daudi cell mixture with effector/target ratio at 2/1. After 24 hours of incubation, the cell mixture was applied to a flow cytometer (iQue3, Sartorius) and the number of live cells of Daudi was counted.

Meanwhile, EM1030-NIC123-N1 and IPH6101 analog (derived from US Patent No. 11,692,039, a multi-specific protein containing a CD123-binding domain, an NKp46-binding domain, and a functional IgG1 Fc, as a control) with serial concentrations (100, 10, 1, 0.1, 0.01 and 0.001 nM) were added to human NK and AML tumor cell mixture including OCI-AML2 (Cobioer, CBP60527) and KG-1 (Immocell, IM-H286) respectively with effector/target ratio at 4/1. After 72 hours of incubation, the cell mixture was applied to a flow cytometer (iQue3, Sartorius) and the number of live cells of AML tumor cells were counted.

To further compare the cytotoxic activities of NK cell-engaging (NKCE) with T cell-engaging (TCE) therapies, EM1030-NIC123-N1, was tested at various concentrations (100, 10, 1, 0.1, 0.01, and 0.001 nM) in individual incubations with human NK cells and each of the four AML tumor cell lines: OCI-AML2 (Cobioer, CBP60527) , KG-1 (Immocell, IM-H286) , THP-1 (ATCC, TIB-202) , and Molm-13 (Immocell, IM-H295) , with an effector-to-target ratio of 4: 1 for each combination. The reference molecule MGD024 analog (US Patent No. 11, 685, 781, a multi-specific protein including a CD123-binding domain and a CD3e-binding domain) was aslo tested following the same procedure except that human T cell was used instead of NK cell therein. After a 72-hour incubation period, the cell mixtures were analyzed using a flow cytometer (iQue3, Sartorius) to determine the number of viable AML tumor cells.

Similarly, the comparison of the cytotoxic activities between NK cell-engaging (NKCE) and monoclonal antibody therapies was also performed, and talacotuzumab (US Patent NO. 8,569,461) , a monoclonal antibody targeting CD123, was used as a control. EM1030-NIC123-N1 and talacotuzumab were tested at various concentrations (100, 10, 1, 0.1, 0.01, and 0.001 nM) in individual incubations with human NK cells and each of the four AML tumor cell lines: OCI-AML2 (Cobioer, CBP60527) , KG-1 (Immocell, IM-H286) , THP-1 (ATCC, TIB-202) , and Molm-13 (Immocell, IM-H295) , with an effector-to-target ratio of 4: 1 for each combination. After a 24-hour incubation period, the cell mixtures were analyzed using a flow cytometer (iQue3, Sartorius) to determine the number of viable AML tumor cells.

The percentage of specific lysis was determined as following:

%Lysis=100%x (1-cell count of sample wells/cell count of untreated wells) Calculated lysis percentages were plotted against test articles concentration with GraphPad Prism software.

As shown in Figure 14A-D, EM1030-NIH-N11, -NIT-N30, -NIA-N3 and -NIC-N3 induced NK cell cytotoxicity towards tumor cells, and their potency was dramatically higher than the corresponding no-IL15v controls.

Figure 14E exhibits that T5-5 induced NK cell cytotoxicity towards tumor cells and its potency was comparable with IPH6501 analog.

Figures 14F and 14G demonstrate enhanced cytotoxic activity of EM1030-NIC123-N1 compared to IPH6101 analog, exhibiting an S-shaped dose-response curve.

Figure 14H shows comparable cytotoxic potency of EM1030-NIC123-N1 to that of MGD024 analog.

Figure 14I indicates that the potency of EM1030-NIC123-N1 is not affected by the CD64 expression of AML cells, demonstrating potent cytotoxicity; meanwhile, the parental anti-CD123 mab has NK cytotoxicity suppressed, depending on CD64 expression of tumor cells.

6.3.2 In vivo anti-tumor efficacy

In vivo anti-tumor growth effect by EM1030-NIH-N11 was evaluated in B16F10-HER2 syngeneic model in B6-hNKp46 knock-in mice (Biocytogen) .

Briefly, B16F10-HER2 tumor cells (generated in-house by transducing human Her2 expression gene into B16F10 cells (ATCC, CRL-6475) ) were routinely cultured for at least two passages before transplantation. On inoculation day, 5x10⁵ B16F10-HER2 tumor cells in 0.1mL DPBS was injected subcutaneously (sc) into right flank of B6-hNKp46 mouse (female) using a 26-gauge springe. Mice with tumor volume of 30-80 mm³ were selected and randomized into three groups (day 0, D0) for treated with EM1030-NIH-N11 (10 mg/kg, 7 mice/group) , EM1030-NIH-N16 (8 mg/kg, as no-IL15v control, 6 mice/group) or vehicle control (DPBS, Basal Media, cat#B210KJ) (7 mice/group) on D0, D5, D9 for three times. Tumor growth and body weight were measured three times a week. Tumor volume was calculated based on dimensions of the tumor using the following formula:

Tumor volume = (length × width²) /2.

The toxicity was assessed by measuring weight loss over time in all animals. Animals observed body weight loss of 20%or above were euthanized.

Figure 15A demonstrates EM1030-NIH-N11 treatment resulted in significant tumor growth inhibition in comparison to vehicle control and EM1030-NIH-N16 treatment group, while there was no difference between vehicle control and EM1030-NIH-N16 group. Figure 15B exhibits body weight change during treatment period, indicating EM1030-NIH-N11 and EM1030-NIH-N16 treatment group was tolerated by these mice.

6.4 Cytokine release

EM1030-NIH-N11 and wild-type IL15-N5 were assessed for the risk to induce cytokine release from PBMC.

Briefly, 0.2 M/well of purified PBMC were seeded in 96-well plate and treated with increasing doses of EM1030-NIH-N11 and IL15-N (100, 10 and 1nM) for 3 days at 37℃, 5%CO₂ in incubator. IL-6 and IFNγ released in the medium was then determined by flow cytometry with LEGENDplex^TM HU Th1 Panel kit (Biolegend, #741036) . The data shown in Figure 16 indicates that EM1030-NIH-N1 has less cytokine release risk in comparison to IL15-N5 control.

To further compare the induction of cytokine release of the protein according to the present disclosure with monoclonal antibody, and other comparable NK/T cell engager, EM1030-NIC123-N1, talacotuzumab, MGD024 analog and IPH6101 analog were also assessed for IL-6, IL-10, TNFα and IFNγ released in the medium, and then determined using the same method above. The data shown in Figure 17 indicates that EM1030-NIC123-N1 shows dramatically reduced cytokine release in comparison with T-cell engaging therapy MGD024 targeting CD123.

List of Sequences

Claims

A multi-specific protein that specifically binds to IL-15 receptor, NKp46 and a target antigen, wherein the multi-specific protein comprises at least three antigen binding portions (ABPs) comprising

one ABP that specifically binds to NKp46, a second ABP that specifically binds to IL-15 receptor, and the third ABP that specifically binds to a target antigen;

wherein the ABP binding IL-15 receptor comprises or consists of an IL15/IL15Rαcomplex, optionally an IL15-sushi complex;

wherein said three ABPs are fused to each other with or without a linker, and

optionally, said multi-specific protein further comprises one or two Fc region.
The multi-specific protein according to claim 1, wherein

the ABP specifically binding NKp46 is a Fab, an scFv or a VHH, and the ABP specifically binding a target antigen is a Fab, an scFv, VHH or Fv;

optionally, the ABP binding NKp46 is a Fab, and the ABP binding a target antigen is an scFv, VHH or Fv; or the ABP binding NKp46 is an scFv, VHH or Fv, and the ABP binding a target antigen is a Fab; or

optionally, the ABP binding NKp46 is a Fab, and the ABP binding a target antigen is a VHH or Fv; or the ABP binding NKp46 is an scFv, and the ABP binding a target antigen is a Fab.
The multi-specific protein according to claim 1, wherein the multi-specific protein comprises:

(i) a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide, wherein

the first polypeptide chain comprises, from N-terminus to C-terminus, VL_A-CL-VH_B-CH1-Fc;

the second polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1;

the third polypeptide chain comprises, from N-terminus to C-terminus, VL_B-CL; and

the fourth polypeptide chain comprises, from N-terminus to C-terminus, IL15Rα-linker-IL15-Fc;

wherein antigen A is NKp46 and antigen B is the target antigen, or B is NKp46 and A is the target antigen;

wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, optionally, the Fc of IgG1, for example the Fc of human IgG1;

wherein said four polypeptide chains are capable of associating to form a binding protein having one first Fab specificity binding to A formed by VH_A-CH1: : VL_A-CL pairing, and one second Fab specificity binding to B formed by VH_B-CH1: : VL_B-CL pairing;

(ii) a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide, wherein

the first polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1-linker-IL15-linker-Fc;

the second polypeptide chain comprises, from N-terminus to C-terminus, VL_A-CL;

the third polypeptide chain comprises, from N-terminus to C-terminus, VL_B-CL-linker-IL15Rα-linker-Fc; and

the fourth polypeptide chain comprises, from N-terminus to C-terminus VH_B-CH1; or

wherein antigen A is NKp46 and antigen B is the target antigen, or B is NKp46 and A is the target antigen;

wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, optionally, the Fc of IgG1, for example, Fc of human IgG1;

wherein said four polypeptide chains are capable of associating to form a binding protein having one first Fab specificity binding to A formed by VH_A-CH1: : VL_A-CL pairing, and one second Fab specificity binding to B formed by VH_B-CH1: : VL_B-CL pairing;

(iii) a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide, wherein

the first polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1-Fc;

the second polypeptide chain comprises, from N-terminus to C-terminus, VL_A-CL;

the third polypeptide chain comprises, from N-terminus to C-terminus, VL_B-linker-IL15; and

the fourth polypeptide chain comprises, from N-terminus to C-terminus VH_B-linker-IL15Rα-linker-Fc;

optionally, wherein IL15Rα and IL15 form a complex by an interchain-di-sulfide bridge;

wherein antigen A is NKp46 and antigen B is the target antigen, or B is NKp46 and A is the target antigen;

wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, optionally, the Fc of IgG1, for example, Fc of human IgG1;

wherein said four polypeptide chains are capable of associating to form a binding protein having one Fab specificity binding to A formed by VH_A-CH1: : VL_A-CL pairing, and one Fv specificity binding to B formed by VH_B: : VL_B pairing;

(iv) a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein

the first polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1-Fc-linker-IL15Rα;

the second polypeptide chain comprises, from N-terminus to C-terminus, VL_A-CL; and

the third polypeptide chain comprises, from N-terminus to C-terminus, ABP_B-linker-Fc-linker-IL15, or ABP_B-linker-Fc-IL15;

optionally, said Fc is effector function silenced or competent;

optionally, ABP_B is an scFv or VHH that binds to antigen B;

wherein antigen A is NKp46 and antigen B is the target antigen, or B is NKp46 and A is the target antigen;

wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, optionally, the Fc of IgG1, for example, the Fc of human IgG1; scFv is a single-chain variable fragement; and VHH is a single heavy chain variable domain;

wherein said three polypeptide chains are capable of associating to form a binding protein having one Fab specificity binding to A formed by VH_A-CH1: : VL_A-CL pairing, and one scFv_B or VHH_B specificity binding to B;

or

(v) one first polypeptide chain, two second polypeptide chains, and one third polypeptide chain, wherein

the first polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1-Fc-linker-scFv_B;

the second polypeptide chain comprises, from N-terminus to C-terminus, VL_A-CL; and

the third polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1-Fc-IL15-linker-IL15Rα;

wherein antigen A is NKp46 and antigen B is the target antigen, or B is NKp46 and A is the target antigen;

wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, optionally, the Fc of IgG1, for example the Fc of human IgG1; and scFv is a single-chain variable fragement;

wherein said four polypeptide chains are capable of associating to form a binding protein having two Fabs specificity binding to A formed by VH_A-CH1: : VL_A-CL pairing, and one scFv_B specificity binding to B;

wherein in (i) to (v) ,

optionally, the linkers in each protein can be identical or not;

optionally, said IL15Rα is a sushi domain of IL-15 receptor alpha;

optionally, said scFv_B comprises or consists of, from N-terminus to C-terminus, VH_B-linker-VL_B, or VL_B-linker-VH_B.
The multi-specific protein according to any one of claims 1 to 3, wherein the ABP specifically binding to NKp46 comprises

CDR-H1, CDR-H2 and CDR-H3 and CDR-L1, CDR-L2 and CDR-L3, wherein,

the CDR-H1 comprises or consists of the amino acid sequence of SEQ ID NO: 6; the CDR-H2 comprises or consists of the amino acid sequence of SEQ ID NO: 7 or 16; the CDR-H3 comprises or consists of the amino acid sequence of SEQ ID NO: 8; the CDR-L1 comprises or consists of the amino acid sequence of SEQ ID NO: 10; the CDR-L2 comprises or consists of the amino acid sequence of SEQ ID NO: 11; the CDR-L3 comprises or consists of the amino acid sequence of SEQ ID NO: 12;

optionally, said CDR-Hs and CDR-Ls are determined according to Kabat scheme.
The multi-specific protein according to any one of claims 1 to 4, wherein the ABP specifically binding to NKp46 comprises a pair of heavy chain variable domain VH and light chain variable domain VL selected from the following pairs of VH and VL:
The multi-specific protein according to any one of claims 1 to 5, wherein the ABP specifically binding to IL-15 receptor comprises IL15 and sushi domain, forming an IL15-sushi complex, wherein IL15 is a wild type IL15 or a variant comprising one or more amino acid substitutions leading to a reduced IL-15 potency;

optionally, wherein said amino acid substitutions are selected from the group consisting of D61A, E64A, N65A, I68A, L69A, N72A, N65E, N65S, N65V and a combination thereof; optionally, said substitution are selected from N65A, L69A or N65A+L69A.
The multi-specific protein according to any one of claims 1 to 6, wherein the IL15 comprises or consists of

(i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NOs: 40, 42 to 48; or

(ii) an amino acid as set forth in any one of SEQ ID NOs: 40, 42 to 48.
The multi-specific protein according to any one of claims 3 to 7, wherein the sushi domain comprises or consists of

(i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 50; or

(ii) an amino acid sequence as set forth in SEQ ID NO: 50.
The multi-specific protein according to any one of claims 3 to 8, wherein each of IL-15 and sushi domain is engineered to contain a cysteine substitution, forming a disulfide bond in the IL15-sushi complex;

optionally, IL15 comprises an amino acid substitution L52C, and sushi domain contains an amino acid substitution S40C;

optionally, IL15 comprises or consists of an amino acid sequence of SEQ ID NO: 51; and the sushi domain comprises or consists of an amino acid sequence of SEQ ID NO: 52.
The multi-specific protein according to any one of claims 1 to 9, wherein the linkers in each protein, identically or not, are independently selected from the group consisting of (GGGGS) ₁ (SEQ ID NO: 54) , (GGGGS) ₂ (SEQ ID NO: 55) , (GGGGS) ₃ (SEQ ID NO: 56) , (GGGGS) ₄ (SEQ ID NO: 57) , (GGGGS) ₆ (SEQ ID NO: 58) , GSSSS, (GSSSS) ₂, (GSSSS) ₃, or (GSSSS) ₃GS.
The multi-specific protein according to any one of claims 3 to 10, wherein the two Fc regions comprise amino acid substitutions, thereby forming Fc heterodimer, optionally,

(i) one Fc region comprises the amino acid substitutions L234A + L235A, L351K and T366K; and the other Fc region comprises the amino acid substitutions L234A +L235A, L351D and L368E (numbering according to the EU index) ;

(ii) one Fc region comprises the amino acid substitutions Y349C + T366S + L368A + Y407V; and the other Fc region comprises the amino acid substitutions S354C +T366W (numbering according to the EU index) ,

(iii) one Fc region comprises the amino acid substitutions L234A + L235A, Y349C, T366S + L368A + Y407V, and D399K + E356K; and the other Fc region comprises the amino acid substitutions L234A + L235A, S354C, T366W, and K409D + K392D (numbering according to the EU index) ; or

(iii) one Fc region comprises the amino acid substitutions S239D + A330L + I332E, Y349C, and T366S + L368A + Y407V; and the other Fc region comprises the amino acid substitutions S239D + A330L + I332E, S354C, and T366W (numbering according to the EU index) .
The multi-specific protein according to any one of claims 1 to 11, wherein the target antigen is a tumor or cancer antigen, e.g., a tumor-associated antigen, optionally, the antigen is selected from Her2, Trop2, LIV-1, ALPP, CD20, CD123 and CD228.
The multi-specific protein according to any one of claims 3 to 12, wherein said scFv in said protein contains one or more cysteine substitutions, forming a disulfide bond in the scFv chain;

optionally, the VL in the scFv contains the amino acid substitution Q100C, and the VH in the scFv contains the amino acid substitution G44C (numbering according to the EU index) .
An anti-NKp46 antibody or antigen-binding fragment thereof, comprising CDR-H1, CDR-H2 and CDR-H3 and CDR-L1, CDR-L2 and CDR-L3, wherein

the CDR-H1 comprises or consists of the amino acid sequence of SEQ ID NO: 6; the CDR-H2 comprises or consists of the amino acid sequence of SEQ ID NO: 16 or 7; the CDR-H3 comprises or consists of the amino acid sequence of SEQ ID NO: 8; the CDR-L1 comprises or consists of the amino acid sequence of SEQ ID NO: 10; the CDR-L2 comprises or consists of the amino acid sequence of SEQ ID NO: 11; the CDR-L3 comprises or consists of the amino acid sequence of SEQ ID NO: 12;

optionally, the CDR-Hs and the CDR-Ls are determined according to Kabat scheme.
The antibody or antigen-binding fragment thereof according to claim 14, comprising a heavy chain variable region VH, wherein the heavy chain variable region comprises or consists of

(i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to an amino acid sequence selected from SEQ ID NOs: 5, 15 and 17-23; or

(ii) an amino acid sequence selected from SEQ ID NOs: 5, 15 and 17-23.
The antibody or antigen-binding fragment thereof according to claim 14 or 15, comprising a light chain variable region VL, wherein the light chain variable region comprises or consists of

(i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to an amino acid sequence selected from SEQ ID NOs: 9, and 24-27; or

(ii) an amino acid sequence selected from SEQ ID NOs: 9, and 24-27.
The antibody or antigen-binding fragment thereof according to claim 14, comprising a heavy chain variable region VH and a light chain variable region VL, wherein,

the VH comprises or consists of the amino acid sequence set forth in SEQ ID NOs: 5, 15 and 17-23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%identity thereto; and

the VL comprises or consists of the amino acid sequence set forth in SEQ ID NOs: 9, and 24-27 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%identity thereto.
The antibody or antigen-binding fragment thereof according to claim 14, comprising a pair of heavy chain variable region VH and light chain variable region VL selected from the group consisting of:
The antibody or antigen-binding fragment thereof according to any one of claims 14 to 18, further comprising a heavy chain constant region CH, e.g., a heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, optionally a heavy chain constant region of IgG1.
The antibody or antigen-binding fragment thereof according to any one of claims 14 to 19, further comprising a light chain constant region CL, e.g., a lambda or kappa light chain constant region, optionally a kappa light chain constant region.
The antibody or antigen-binding fragment thereof according to any one of claims 14 to 20, wherein the antibody is a humanized antibody or a chimeric antibody.
The antibody or antigen-binding fragment thereof according to any one of claims 14 to 21, wherein the antibody is a monoclonal antibody.
The antibody or antigen-binding fragment thereof according to any one of claims 14 to 22 wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, single chain antibodies (e.g., scFv) , (Fab') 2, single domain antibodies such as VHH, dAb (domain antibody) , or linear antibodies.
A nucleic acid molecule encoding any chain of the antibody or antigen-binding fragment thereof according to any one of claims 14 to 23, or any chain of the multi-specific protein according to any one of claims 1 to 12.
An expression vector comprising the nucleic acid molecule according to claim 24, preferably the expression vector is pcDNA, such as pcDNA3.1.
A host cell comprising the nucleic acid molecule according to claim 24, or the expression vector according to claim 25, preferably the host cell is prokaryotic or eukaryotic, such as HEK293 cells, or CHO cells, such as 293E cells or CHO-K1 cells.
A method of making the antibody or antigen-binding fragment thereof according to any one of claims 14 to 23, or the multi-specific protein according to any one of claims 1-12, wherein the method comprises culturing a host cell comprising the nucleic acid molecule according to claim 24 or the expression vector according to claim 25 under conditions suitable for chain expression of the antibody or protein, and optionally, recovering the antibody or protein from the host cell (or host cell culture medium) .
An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 14 to 23 or the multi-specific protein according to any one of claims 1 to 12.
A pharmaceutical composition or medicament or formulation comprising the antibody or antigen-binding fragment thereof according to any one of claims 14 to 23, or the multi-specific protein according to any one of claims 1 to 12, or the immunoconjugate of claim 28, and optionally a pharmaceutical acceptable supplementary material.
A pharmaceutical combination comprising the antibody or antigen-binding fragment thereof according to any one of claims 14 to 23 or the multi-specific protein according to any one of claims 1 to 12, or the immunoconjugate of claim 28, and one or more additional ingredient useful in the treatment of various diseases, e.g., a prophylactic and/or therapeutic agent, such as a detection agent, an anti-tumor drug, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an antibody of different specificity or functional fragment thereof, a small molecule drug, an immunosuppressant or a detectable label or reporter.
A method of preventing or treating a disorder or disease in a subject in need thereof, comprising administering to the subject an effective amount of the multi-specific protein according to any one of claims 1 to 12; the immunoconjugate of claim 28; the pharmaceutical composition or formulation of claim 29; or the pharmaceutical combination of claim 30.
The method according to claim 31, wherein the disorder or disease is a cancer, optionally, solid cancer or hematopoietic cancer, e.g., a cancer of a tissue selected from the group consisting of colon, rectum, cervix, oropharynx, nasopharynx, liver, stomach, head and neck, oral cavity, oesophagus, lip, mouth, tongue, tonsil, nose, throat, salivary gland, sinus, pharynx, larynx, prostate, lung, bladder, skin, kidney, ovary and mesothelium.
The method according to claim 31 or 32, wherein the disorder or disease is a cancer selected from the group consisting of melanoma, metastatic melanoma, renal cell carcinoma, ovarian cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, brain cancer, head and neck cancer, breast cancer (including three-negative breast cancer) , colon cancer, rectal cancer, colorectal cancer, skin cancer, throat cancer, uterine cancer, cervical carcinoma, hepatocellular carcinoma, prostate cancer, pancreatic cancer, gastric cancer, chronic lymphocytic leukemia (CLL) , multiple myeloma, lymphoma and bladder cancer.
The method according to any one of claims 31 to 33, wherein the method further comprises administration in combination with one or more therapies, such as surgery or radiotherapy, or additional agents useful in the treatment of various diseases, e.g., a prophylactic and/or therapeutic agent, such as a detection agent, an anti-tumor drug, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an antibody of different specificity or functional fragment thereof, a small molecule drug, an immunosuppressant or a detectable label or reporter.
A method for detecting the presence of NKp46 in a biological sample comprising

(i) contacting a biological sample with the antibody or antigen-binding fragment thereof according to any one of claims 14 to 23 or the multi-specific protein according to any one of claims 1-12 under the conditions that allow binding to NKp46; and

(ii) detecting whether a complex is formed between the antibody or the multi-specific protein and NKp46;

wherein the formation of a complex indicates the presence of NKp46.