Use of CD123 NK cell adapters for treating aml and related disorders

Abstract

Provided herein are methods for treating or preventing leukemia and myelodysplasia disorders comprising a multifunctional binding protein comprising a first antigen-binding domain and a second antigen-binding domain and all or part of an immunoglobulin Fc region or variant thereof wherein the first antigen-binding domain specifically binds to CD123, and the second antigen-binding domain specifically binds to CD123, and all or part of an immunoglobulin Fc region or variant thereof. And the second antigen-binding domain specifically binds to human NKp46, and wherein all or part of the immunoglobulin Fc region or variant thereof binds to a human Fc-gamma receptor.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to the following U.S. provisional patent application serial numbers: 63 / 461,480, filed April 24, 2023; 63 / 464,404, filed May 5, 2023; 63 / 465,218, filed May 9, 2023; 63 / 468,634, filed May 24, 2023; and 63 / 526,661, filed July 13, 2023. The contents of 63 / 528,509, filed on 24 July 2023; 63 / 529,919, filed on 31 July 2023; 63 / 538,210, filed on 13 September 2023; 63 / 597,622, filed on 9 November 2023; and 63 / 559,491, filed on 29 February 2024, are hereby incorporated in their entirety by reference. Background Technology

[0003] Acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), and myelodysplastic syndromes (MDS) are heterogeneous clonal neoplastic diseases believed to be caused by a subset of leukemia stem cells that are often resistant to conventional chemotherapy and may further lead to disease relapse. Patients diagnosed with relapsed or refractory HR-MDS face a poor median overall survival (OS) after unsuccessful treatment with hypomethylating agents (HMAs).

[0004] Blastoblastic plasmacytoid dendritic cell tumor (BPDCN) is a rare, aggressive hematologic malignancy caused by plasmacytoid dendritic cell progeny. Several different nomenclatures were used to describe BPDCN in the past until the 2008 World Health Organization (WHO) classification described it as an entity under the family of acute myeloid leukemia (AML) and related tumors. In a subsequent WHO reclassification, it was given its own separate category under myeloid neoplasms, reflecting its unique pathobiology. BPDCN is typically presented as cutaneous lesions with or without bone marrow involvement, lymphadenopathy, splenomegaly, cytopenia, and sometimes extramedullary involvement.

[0005] Natural killer (NK) cell lines are lymphocyte subsets involved in unconventional immunity. NK cells provide an effective immune surveillance mechanism by eliminating unwanted cells, such as tumor-infected or virus-infected cells. Characteristic and biological properties of NK cells include the expression of surface antigens (including CD16, CD56, and / or CD57); the absence of α / β or γ / δ TCR complexes on the cell surface; the ability to bind to and kill cells (especially those that do not express "self" MHC / HLA antigens) in an MHC-unrestricted manner by activating specific cell-lysing enzymes; the ability to kill tumor cells or other diseased cells expressing ligands of the NK activation receptor; and the ability to release protein molecules that stimulate an immune response (called cytokines).

[0006] Nevertheless, there remains an urgent need for treatment options for patients who do not meet the criteria for standard treatment or who have exhausted standard treatment options. Summary of the Invention

[0007] In one aspect, this article provides a method for treating or preventing leukemia or myelodysplastic syndrome in a subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46, wherein the binding protein is administered to the subject at a dose of at least 3 µg / kg.

[0008] On the other hand, this article provides a method for treating or preventing leukemia or myelodysplastic syndrome in a subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46, wherein the first antigen-binding domain comprises:

[0009] The heavy chain variable domain (VH1) comprises CDR-H1, H2, and H3 corresponding to amino acid sequences of SEQ ID NO: 1, 2, and 3, or corresponding to amino acid sequences of SEQ ID NO: 4, 5, and 5, respectively; and

[0010] The light chain variable domain (VL1) contains CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 7, 8 and 9, respectively, or corresponding to the amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively.

[0011] The binding protein was administered to the subject at a dose of at least 3 µg / kg.

[0012] In some embodiments, the dose is between about 3 µg / kg and about 6000 µg / kg. In some embodiments, the dose is between about 3 µg / kg and about 30 µg / kg. In some embodiments, the dose is between about 10 µg / kg and 100 µg / kg. In some embodiments, the dose is between about 30 µg / kg and 100 µg / kg. In some embodiments, the dose is between about 13 µg / kg and about 130 µg / kg. In some embodiments, the dose is between about 15 µg / kg and about 150 µg / kg. In some embodiments, the dose is between about 20 µg / kg and about 200 µg / kg. In some embodiments, the dose is between about 30 µg / kg and about 300 µg / kg. In some embodiments, the dose is between about 40 µg / kg and about 400 µg / kg. In some embodiments, the dose is between about 45 µg / kg and about 450 µg / kg. In some embodiments, the dose is between about 60 µg / kg and about 600 µg / kg. In some embodiments, the dose is between about 100 µg / kg and about 1000 µg / kg. In some embodiments, the dose is between about 300 µg / kg and about 1000 µg / kg. In some embodiments, the dose is between 400 µg / kg and about 1300 µg / kg. In some embodiments, the dose is between about 450 µg / kg and about 1500 µg / kg. In some embodiments, the dose is between about 600 µg / kg and about 1000 µg / kg. In some embodiments, the dose is between about 600 µg / kg and about 1500 µg / kg. In some embodiments, the dose is between about 600 µg / kg and about 2000 µg / kg. In some embodiments, the dose is between about 1000 µg / kg and about 3000 µg / kg. In some embodiments, the dose is between about 1300 µg / kg and about 3000 µg / kg. In some embodiments, the dose is between about 1500 µg / kg and about 3000 µg / kg. In some embodiments, the dose is between about 2000 µg / kg and about 3000 µg / kg.

[0013] In some embodiments, the dose is about 3 µg / kg. In some embodiments, the dose is about 10 µg / kg. In some embodiments, the dose is about 13 µg / kg. In some embodiments, the dose is about 15 µg / kg. In some embodiments, the dose is about 20 µg / kg. In some embodiments, the dose is about 30 µg / kg. In some embodiments, the dose is about 40 µg / kg. In some embodiments, the dose is about 45 µg / kg. In some embodiments, the dose is about 50 µg / kg. In some embodiments, the dose is about 60 µg / kg. In some embodiments, the dose is about 100 µg / kg. In some embodiments, the dose is about 130 µg / kg. In some embodiments, the dose is about 150 µg / kg. In some embodiments, the dose is about 200 µg / kg. In some embodiments, the dose is about 300 µg / kg. In some embodiments, the dose is about 400 µg / kg. In some embodiments, the dose is about 450 µg / kg. In some embodiments, the dose is about 500 µg / kg. In some embodiments, the dose is about 600 µg / kg. In some embodiments, the dose is about 750 µg / kg. In some embodiments, the dose is about 1000 µg / kg. In some embodiments, the dose is about 1300 µg / kg. In some embodiments, the dose is about 1500 µg / kg. In some embodiments, the dose is about 2000 µg / kg. In some embodiments, the dose is about 3000 µg / kg. In some embodiments, the dose is about 4000 µg / kg. In some embodiments, the dose is about 4500 µg / kg. In some embodiments, the dose is about 6000 µg / kg.

[0014] In some embodiments, the binding protein is administered to the subject intravenously, subcutaneously, intraperitoneally, or intramuscularly. In some embodiments, the binding protein is administered to the subject intravenously.

[0015] In some embodiments, the binding protein is administered to the subject for at least one cycle. In some embodiments, the at least one cycle is approximately 28 days. In some embodiments, the binding protein is administered to the subject for at least two cycles. In some embodiments, the binding protein is administered to the subject for three or more cycles.

[0016] In some embodiments, the first antigen-binding domain having binding specificity to CD123 includes:

[0017] The heavy chain variable domain (VH1) comprises CDR-H1, H2, and H3 corresponding to amino acid sequences of SEQ ID NO: 1, 2, and 3, or corresponding to amino acid sequences of SEQ ID NO: 4, 5, and 6, respectively; and

[0018] The light chain variable domain (VL1) contains CDR-L1, L2 and L3 corresponding to amino acid sequences of SEQ ID NO: 7, 8 and 9, or amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively.

[0019] In some embodiments, VH1 contains at least about 90% of the amino acid sequence identical to that of SEQ ID NO: 41, and wherein VL1 contains at least about 90% of the amino acid sequence identical to that of SEQ ID NO: 43; or VH1 contains at least about 90% of the amino acid sequence identical to that of SEQ ID NO: 42, and wherein VL1 contains at least about 90% of the amino acid sequence identical to that of SEQ ID NO: 44.

[0020] In some embodiments, the VH1 comprises the amino acid sequence of SEQ ID NO: 41, and the VL1 comprises the amino acid sequence of SEQ ID NO: 43; or the VH1 comprises the amino acid sequence of SEQ ID NO: 42, and the VL1 comprises the amino acid sequence of SEQ ID NO: 44.

[0021] In some embodiments, the second antigen-binding domain having binding specificity to NKp46 comprises:

[0022] a. The second heavy chain variable domain (VH2) contains CDRs H1, H2, and H3 corresponding to the following amino acid sequences:

[0023] i. Specifically, SEQ ID NO: 13, 14 and 15;

[0024] ii. Specifically, SEQ ID NO: 16, 17, and 18;

[0025] iii. Specifically, SEQ ID NO: 19, 20, and 21;

[0026] iv. respectively, SEQ ID NO: 22, 23 and 24; or

[0027] v. respectively, SEQ ID NO: 16, 25 and 26; and

[0028] b. The second light chain variable domain (VL2) contains CDRs-L1, L2, and L3 corresponding to the following amino acid sequences:

[0029] i. Specifically, SEQ ID NO: 27, 28, and 29;

[0030] ii. Specifically, SEQ ID NO: 30, 31 and 32;

[0031] iii. Specifically, SEQ ID NO: 33, 34 and 35;

[0032] iv. respectively, SEQ ID NO: 36, 37 and 38; or

[0033] v. respectively, SEQ ID NO: 39, 31 and 40.

[0034] In some embodiments, VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 45, and wherein VL1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 53; VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 46, and wherein VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 54; VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 47, and wherein VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 55; VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 48, and wherein VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 56; VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and wherein VL ... The amino acid sequence of SEQ ID NO: 57 is at least about 90% identical to the amino acid sequence of SEQ ID NO: 50; the VH2 contains at least about 90% identical to the amino acid sequence of SEQ ID NO: 58; the VH2 contains at least about 90% identical to the amino acid sequence of SEQ ID NO: 51; the VL2 contains at least about 90% identical to the amino acid sequence of SEQ ID NO: 59; or the VH2 contains at least about 90% identical to the amino acid sequence of SEQ ID NO: 52; the VL2 contains at least about 90% identical to the amino acid sequence of SEQ ID NO: 60.

[0035] In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 45, and wherein VL1 comprises the amino acid sequence of SEQ ID NO: 53; VH2 comprises the amino acid sequence of SEQ ID NO: 46, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 54; VH2 comprises the amino acid sequence of SEQ ID NO: 47, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 55; VH2 comprises the amino acid sequence of SEQ ID NO: 48, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 56; VH2 comprises the amino acid sequence of SEQ ID NO: 49, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 57; VH2 comprises the amino acid sequence of SEQ ID NO: 50, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 58; VH2 comprises the amino acid sequence of SEQ ID NO: 51, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 59; or VH2 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of SEQ ID NO: 52, wherein the VL2 comprises the amino acid sequence of SEQ ID NO: 60.

[0036] In some embodiments, the binding protein comprises three polypeptide chains (I), (II), and (III) that form two ABDs, as defined below:

[0037] V 1A - C 1A - Hinge 1 - (C H 2-C H 3) A (I)

[0038] V 1B - C 1B - Hinge 2 - (C H 2-C H 3) B - L1 - V 2A - C 2A - Hinge 3(II)

[0039] V 2B - C 2B (III)

[0040] in:

[0041] V 1A and V 1B Forming binding pairs V1 (V H1 / VL1 );

[0042] V 2A and V 2B Forming binding pairs V2 (V H2 / V L2 );

[0043] C 1A and C 1B Forming a pair of C1 (C H 1 / C L ), and C 2A and C 2B Formation of C2 (C H 1 / C L ), where C H 1 represents the immunoglobulin heavy chain constant domain 1, and C L It is the constant structural domain of the immunoglobulin light chain;

[0044] Hinge 1, hinge 2 and hinge 3 may be the same or different, and correspond to all or part of the immunoglobulin hinge region;

[0045] (C H 2-C H 3) A and (C) H 2-C H 3) B For the same or different, and containing the immunoglobulin heavy chain constant domain 2 (C H 2) and immunoglobulin heavy chain constant domain 3 (C H 3);

[0046] L1 is an amino acid linker.

[0047] In some implementation schemes, C 1B For immunoglobulin heavy chain constant domain 1 (C H 1);

[0048] C 2A For immunoglobulin heavy chain constant domain 1 (C H 1);

[0049] C L Corresponding to the immunoglobulin κ light chain constant domain (C κ );

[0050] (C H 2-C H 3) A The amino acid sequence corresponding to SEQ ID NO: 69;

[0051] (C H 2-CH 3) B The amino acid sequence corresponding to SEQ ID NO: 70;

[0052] Hinge 1 corresponds to the amino acid sequence of SEQ ID NO: 74;

[0053] Hinge 2 corresponds to the amino acid sequence of SEQ ID NO: 75;

[0054] Hinge 3 corresponds to the amino acid sequence of SEQ ID NO: 77;

[0055] L1 corresponds to the amino acid sequence of SEQ ID NO: 76.

[0056] In some implementations, residue N297 of the Fc region or its variants, according to the EU number, contains N-linked glycosylation.

[0057] In some embodiments, all or part of the Fc region or a variant thereof binds to the human CD16A (FcγRIII) polypeptide.

[0058] In some embodiments, at least two polypeptide chains are linked by at least one disulfide bond. In some embodiments, polypeptide chain (I) and polypeptide chain (II) are linked by a C 1A At least one disulfide bond is connected between hinge 2 and the polypeptide chain (II) and the polypeptide chain (III) via hinge 3 to C. 2B At least one disulfide bond is linked between them. In some embodiments, V 1A For V L1 And V 1B For V H1 In some implementations, V 2A For V H2 And V 2B For V L2 .

[0059] In some implementation schemes, V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2The CDR-H1 contains the amino acid sequence of SEQ ID NO: 13; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 14; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 15; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 27; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 28; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 29.

[0060] V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 17; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 18; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO:30; the CDR-L2 contains the amino acid sequence of SEQ ID NO:31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:32.

[0061] V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 19; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 20; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 21; V L2The CDR-L1 contains the amino acid sequence of SEQ ID NO:33; the CDR-L2 contains the amino acid sequence of SEQ ID NO:34; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:35.

[0062] V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 22; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 23; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 24; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO:36; the CDR-L2 contains the amino acid sequence of SEQ ID NO:37; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:38.

[0063] V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 25; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 26; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO:39; the CDR-L2 contains the amino acid sequence of SEQ ID NO:31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:40.

[0064] V H1The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 13; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 14; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 15; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 27; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 28; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 29.

[0065] V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 17; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 18; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 30; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 32.

[0066] V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 19; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 20; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 21; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 33; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 34; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 35.

[0067] V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 22; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 23; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 24; V L2 The CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; the CDR-L2 containing the amino acid sequence of SEQ ID NO: 37; the CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; or

[0068] V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 25; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 26; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 39; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 40.

[0069] In some implementations, the V H1 and the V L1 The amino acid sequences corresponding to SEQ ID NO: 41 and 43, or the amino acid sequences corresponding to SEQ ID NO: 42 and 44, respectively; and / or V H2 and V L2 Corresponding to

[0070] The amino acid sequences of SEQ ID NO: 45 and 53, respectively;

[0071] The amino acid sequences of SEQ ID NO: 46 and 54, respectively;

[0072] The amino acid sequences of SEQ ID NO: 47 and 55, respectively;

[0073] The amino acid sequences of SEQ ID NO: 48 and 56, respectively;

[0074] The amino acid sequences of SEQ ID NO: 49 and 57, respectively;

[0075] The amino acid sequences of SEQ ID NO: 50 and 58, respectively;

[0076] Specifically, the amino acid sequences of SEQ ID NO: 51 and 59; or

[0077] The amino acid sequences of SEQ ID NO: 52 and 60, respectively.

[0078] In some implementation schemes, V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 45; V L2 Contains the amino acid sequence of SEQ ID NO: 53; V H1 Contains the amino acid sequence of SEQ ID NO: 41; VL1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 46; V L2 Contains the amino acid sequence of SEQ ID NO: 54; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 47; V L2 Contains the amino acid sequence of SEQ ID NO: 55; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 48; V L2 Contains the amino acid sequence of SEQ ID NO: 56; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 49; VL2 contains the amino acid sequence of SEQ ID NO: 57; V H1 Contains the amino acid sequence of SEQ ID NO:41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 50; V L2 Contains the amino acid sequence of SEQ ID NO: 58; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 51; V L2 Contains the amino acid sequence of SEQ ID NO: 59; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 52; VL2 contains the amino acid sequence of SEQ ID NO: 60; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1Contains the amino acid sequence of SEQ ID NO: 44; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 45; VL2 contains the amino acid sequence of SEQ ID NO: 53; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 46; V L2 The amino acid sequence containing SEQ ID NO:54; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 47; V L2 Contains the amino acid sequence of SEQ ID NO: 55; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 48; V L2 Contains the amino acid sequence of SEQ ID NO: 56; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 49; V L2 Contains the amino acid sequence of SEQ ID NO: 57; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 50; V L2 Contains the amino acid sequence of SEQ ID NO: 58; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO:51; V L2 Contains the amino acid sequence of SEQ ID NO: 59; or V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 52; V L2 It contains the amino acid sequence of SEQ ID NO: 60.

[0079] In some embodiments, polypeptide (I) consists of the amino acid sequence of SEQ ID NO: 64; polypeptide (II) consists of the amino acid sequence of SEQ ID NO: 65; and polypeptide (III) consists of the amino acid sequence of SEQ ID NO: 66.

[0080] In some embodiments, the leukemia is acute myeloid leukemia (AML). In some embodiments, the AML is relapsed or refractory. In some embodiments, the leukemia is B-cell acute lymphoblastic leukemia (B-ALL). In some embodiments, the blood disorder or disorder is myelodysplastic syndrome. In some embodiments, the myelodysplastic syndrome is high-risk myelodysplastic syndrome (HR-MDS).

[0081] In one aspect, this article provides a method for treating or preventing leukemia or myelodysplastic syndrome in a subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46.

[0082] The first antigen-binding domain comprises a heavy chain variable domain (VH1) and a light chain variable domain (VL1), wherein:

[0083] The VH1 contains complementarity-determining regions (CDRs) - H1, H2, and H3 corresponding to the amino acid sequences of SEQ ID NO: 1, 2, and 3; and

[0084] The VL1 comprises CDR-L1, L2, and L3 corresponding to the amino acid sequences of SEQ ID NO: 7, 8, and 9; and

[0085] The second antigen-binding domain comprises a heavy chain variable domain (VH2) and a light chain variable domain (VL2), wherein:

[0086] The VH2 contains CDR-H1, H2, and H3 corresponding to the amino acid sequences of SEQ ID NO: 13, 14, and 15; and

[0087] The VL2 comprises CDR-L1, L2, and L3 corresponding to the amino acid sequences of SEQ ID NO: 27, 28, and 29;

[0088] Furthermore, all or part of the Fc region of the immunoglobulin or its variants bind to the human Fc-γ receptor.

[0089] The binding protein was administered to the subject at a dose of at least 3 µg / kg.

[0090] On the other hand, this article provides a method for treating or preventing leukemia or myelodysplastic disorder in a subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123, a second antigen-binding domain having binding specificity to NKp46, and all or part of an immunoglobulin Fc region or a variant thereof that binds to a human Fc-γ receptor, wherein the binding protein comprises:

[0091] A polypeptide (I) consisting of the amino acid sequence of SEQ ID NO: 64;

[0092] A polypeptide (II) consisting of the amino acid sequence of SEQ ID NO: 65; and

[0093] A polypeptide (III) consisting of the amino acid sequence of SEQ ID NO: 66.

[0094] The binding protein was administered to the subject at a dose of at least 3 µg / kg.

[0095] On the other hand, this article provides a method for treating or preventing leukemia or myelodysplastic syndrome in a subject in need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46.

[0096] In one aspect, this article provides a method for treating or preventing leukemia or myelodysplastic syndrome in a subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46, wherein the first antigen-binding domain comprises:

[0097] The heavy chain variable domain (VH1) comprises CDR-H1, H2, and H3 corresponding to amino acid sequences of SEQ ID NO: 1, 2, and 3, or corresponding to amino acid sequences of SEQ ID NO: 4, 5, and 6, respectively; and

[0098] The light chain variable domain (VL1) contains CDR-L1, L2 and L3 corresponding to amino acid sequences of SEQ ID NO: 7, 8 and 9, or amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively.

[0099] In one aspect, this article provides a method for treating or preventing leukemia or hematologic malignancies in pediatric subjects in need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46.

[0100] On the other hand, this article provides a method for treating or preventing blastic plasmacytoid dendritic cell tumors (BPDCN) in a subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46.

[0101] On the other hand, this article provides a method for treating or preventing blastic plasmacytoid dendritic cell tumor (BPDCN) in a subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46, wherein the first antigen-binding domain comprises:

[0102] The heavy chain variable domain (VH1) comprises CDR-H1, H2, and H3 corresponding to amino acid sequences of SEQ ID NO: 1, 2, and 3, or corresponding to amino acid sequences of SEQ ID NO: 4, 5, and 5, respectively; and

[0103] The light chain variable domain (VL1) contains CDR-L1, L2 and L3 corresponding to amino acid sequences of SEQ ID NO: 7, 8 and 9, or amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively.

[0104] In some implementations, the subjects are pediatric subjects.

[0105] In some embodiments, the binding protein is administered to the subject intravenously, subcutaneously, intraperitoneally, or intramuscularly. In some embodiments, the binding protein is administered to the subject intravenously.

[0106] In some embodiments, the binding protein is administered at a dose between about 500 µg / kg and about 12000 µg / kg. In some embodiments, the binding protein is administered at a dose between about 600 µg / kg and about 1000 µg / kg. In some embodiments, the binding protein is administered at a dose between about 600 µg / kg and about 1500 µg / kg. In some embodiments, the dose is about 500 µg / kg. In some embodiments, the dose is about 600 µg / kg. In some embodiments, the dose is about 1000 µg / kg. In some embodiments, the dose is about 1500 µg / kg. In some embodiments, the dose is about 3000 µg / kg. In some embodiments, the dose is about 6000 µg / kg. In some embodiments, the dose is about 12000 µg / kg.

[0107] In some embodiments, the first antigen-binding domain having binding specificity to CD123 comprises: a heavy chain variable domain (VH1) containing CDR-H1, H2, and H3 corresponding to amino acid sequences of SEQ ID NO: 1, 2, and 3, or corresponding to amino acid sequences of SEQ ID NO: 4, 5, and 5, respectively; and

[0108] The light chain variable domain (VL1) contains CDR-L1, L2 and L3 corresponding to amino acid sequences of SEQ ID NO: 7, 8 and 9, or amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively.

[0109] In some embodiments, VH1 contains at least about 90% of the amino acid sequence identical to that of SEQ ID NO: 41, and wherein VL1 contains at least about 90% of the amino acid sequence identical to that of SEQ ID NO: 43; or VH1 contains at least about 90% of the amino acid sequence identical to that of SEQ ID NO: 42, and wherein VL1 contains at least about 90% of the amino acid sequence identical to that of SEQ ID NO: 44.

[0110] In some embodiments, the VH1 comprises the amino acid sequence of SEQ ID NO: 41, and the VL1 comprises the amino acid sequence of SEQ ID NO: 43; or the VH1 comprises the amino acid sequence of SEQ ID NO: 42, and the VL1 comprises the amino acid sequence of SEQ ID NO: 44.

[0111] In some embodiments, the second antigen-binding domain having binding specificity to NKp46 comprises a second heavy chain variable domain (VH2) containing CDR-H1, H2, and H3 corresponding to the following amino acid sequences: respectively, SEQ ID NOs: 13, 14, and 15; respectively, SEQ ID NOs: 16, 17, and 18; respectively, SEQ ID NOs: 19, 20, and 21; respectively, SEQ ID NOs: 22, 23, and 24; or respectively, SEQ ID NOs: 16, 25, and 26; and

[0112] The second light chain variable domain (VL2) comprises CDR-L1, L2, and L3 corresponding to the following amino acid sequences: respectively, SEQ ID NO: 27, 28, and 29; respectively, SEQ ID NO: 30, 31, and 32; respectively, SEQ ID NO: 33, 34, and 35; respectively, SEQ ID NO: 36, 37, and 38; or respectively, SEQ ID NO: 39, 31, and 40.

[0113] In some embodiments, VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 45, and wherein VL1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 53; VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 46, and wherein VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 54; VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 47, and wherein VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 55; VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 48, and wherein VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 56; VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and wherein VL ... The amino acid sequence of SEQ ID NO: 57 is at least about 90% identical to the amino acid sequence of SEQ ID NO: 50; the VH2 contains at least about 90% identical to the amino acid sequence of SEQ ID NO: 58; the VH2 contains at least about 90% identical to the amino acid sequence of SEQ ID NO: 51; the VL2 contains at least about 90% identical to the amino acid sequence of SEQ ID NO: 59; or the VH2 contains at least about 90% identical to the amino acid sequence of SEQ ID NO: 52; the VL2 contains at least about 90% identical to the amino acid sequence of SEQ ID NO: 60.

[0114] In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 45, and wherein VL1 comprises the amino acid sequence of SEQ ID NO: 53; VH2 comprises the amino acid sequence of SEQ ID NO: 46, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 54; VH2 comprises the amino acid sequence of SEQ ID NO: 47, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 55; VH2 comprises the amino acid sequence of SEQ ID NO: 48, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 56; VH2 comprises the amino acid sequence of SEQ ID NO: 49, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 57; VH2 comprises the amino acid sequence of SEQ ID NO: 50, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 58; VH2 comprises the amino acid sequence of SEQ ID NO: 51, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 59; or VH2 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of SEQ ID NO: 52, wherein the VL2 comprises the amino acid sequence of SEQ ID NO: 60.

[0115] In some embodiments, the binding protein comprises three polypeptide chains (I), (II), and (III) that form two ABDs, as defined below:

[0116] V 1A - C 1A - Hinge 1 - (C H 2-C H 3) A (I)

[0117] V 1B - C 1B - Hinge 2 - (C H 2-C H 3) B - L1 - V 2A - C 2A - Hinge 3(II)

[0118] V 2B - C 2B (III)

[0119] in:

[0120] V 1A and V 1B Forming binding pairs V1 (V H1 / VL1 );

[0121] V 2A and V 2B Forming binding pairs V2 (V H2 / V L2 );

[0122] C 1A and C 1B Forming a pair of C1 (C H 1 / C L ), and C 2A and C 2B Formation of C2 (C H 1 / C L ), where C H 1 represents the immunoglobulin heavy chain constant domain 1, and C L It is the constant structural domain of the immunoglobulin light chain;

[0123] Hinge 1, hinge 2 and hinge 3 may be the same or different, and correspond to all or part of the immunoglobulin hinge region;

[0124] (C H 2-C H 3) A and (C) H 2-C H 3) B For the same or different, and containing the immunoglobulin heavy chain constant domain 2 (C H 2) and immunoglobulin heavy chain constant domain 3 (C H 3);

[0125] L1 is an amino acid linker.

[0126] In some implementation schemes, C 1B For immunoglobulin heavy chain constant domain 1 (C H 1);

[0127] C 2A For immunoglobulin heavy chain constant domain 1 (C H 1);

[0128] C L Corresponding to the immunoglobulin κ light chain constant domain (C κ );

[0129] (C H 2-C H 3) A The amino acid sequence corresponding to SEQ ID NO: 69;

[0130] (C H 2-CH 3) B The amino acid sequence corresponding to SEQ ID NO: 70;

[0131] Hinge 1 corresponds to the amino acid sequence of SEQ ID NO: 74;

[0132] Hinge 2 corresponds to the amino acid sequence of SEQ ID NO: 75;

[0133] Hinge 3 corresponds to the amino acid sequence of SEQ ID NO: 77;

[0134] L1 corresponds to the amino acid sequence of SEQ ID NO: 76.

[0135] In some embodiments, residue N297 of the Fc region or a variant thereof, according to EU number, contains an N-linked glycosylation. In some embodiments, all or part of the Fc region or a variant thereof binds to the human CD16A (FcγRIII) polypeptide. In some embodiments, the binding protein comprises at least two polypeptide chains linked by at least one disulfide bond.

[0136] In some embodiments, the polypeptide chain (I) and the polypeptide chain (II) are coupled via C 1A At least one disulfide bond is connected between hinge 2 and the polypeptide chain (II) and the polypeptide chain (III) via hinge 3 to C. 2B At least one disulfide bond connects them.

[0137] In some implementation schemes, V 1A For V L1 And V 1B For V H1 In some implementations, V 2A For V H2 And V 2B For V L2 .

[0138] In some implementation schemes, V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2The CDR-H1 contains the amino acid sequence of SEQ ID NO: 13; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 14; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 15; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 27; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 28; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 29; V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 17; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 18; V L2 CDR-L1 containing the amino acid sequence of SEQ ID NO: 30; CDR-L2 containing the amino acid sequence of SEQ ID NO: 31; CDR-L3 containing the amino acid sequence of SEQ ID NO: 32; V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 19; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 20; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 21; V L2 CDR-L1 containing the amino acid sequence of SEQ ID NO: 33; CDR-L2 containing the amino acid sequence of SEQ ID NO: 34; CDR-L3 containing the amino acid sequence of SEQ ID NO: 35; VH1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 22; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 23; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 24; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 36; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 37; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 38; V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 25; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 26; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 39; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 40; V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 13; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 14; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 15; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 27; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 28; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 29; V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 17; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 18; V L2 CDR-L1 containing the amino acid sequence of SEQ ID NO: 30; CDR-L2 containing the amino acid sequence of SEQ ID NO: 31; CDR-L3 containing the amino acid sequence of SEQ ID NO: 32; V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2The CDR-H1 contains the amino acid sequence of SEQ ID NO: 19; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 20; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 21; V L2 CDR-L1 containing the amino acid sequence of SEQ ID NO: 33; CDR-L2 containing the amino acid sequence of SEQ ID NO: 34; CDR-L3 containing the amino acid sequence of SEQ ID NO: 35; V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 22; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 23; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 24; V L2 The CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; the CDR-L2 containing the amino acid sequence of SEQ ID NO: 37; the CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; or V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 25; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 26; V L2The CDR-L1 contains the amino acid sequence of SEQ ID NO: 39; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 40.

[0139] In some implementation schemes, V H1 and V L1 The amino acid sequences corresponding to SEQ ID NO: 41 and 43, or the amino acid sequences corresponding to SEQ ID NO: 42 and 44, respectively; and / or V H2 and V L2 Corresponding to

[0140] The amino acid sequences of SEQ ID NO: 45 and 53;

[0141] The amino acid sequences of SEQ ID NO: 46 and 54;

[0142] The amino acid sequences of SEQ ID NO: 47 and 55;

[0143] The amino acid sequences of SEQ ID NO: 48 and 56;

[0144] The amino acid sequences of SEQ ID NO: 49 and 57;

[0145] The amino acid sequences of SEQ ID NO: 50 and 58;

[0146] The amino acid sequences of SEQ ID NO: 51 and 59; or

[0147] The amino acid sequences of SEQ ID NO: 52 and 60.

[0148] In some implementation schemes, V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 45; V L2 Contains the amino acid sequence of SEQ ID NO: 53; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 46; V L2 Contains the amino acid sequence of SEQ ID NO: 54; V H1 Contains the amino acid sequence of SEQ ID NO: 41; VL1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 47; V L2 Contains the amino acid sequence of SEQ ID NO: 55; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 48; V L2 Contains the amino acid sequence of SEQ ID NO: 56; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 49; VL2 contains the amino acid sequence of SEQ ID NO: 57; V H1 The amino acid sequence containing SEQ ID NO:41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 50; V L2 Contains the amino acid sequence of SEQ ID NO: 58; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 51; V L2 Contains the amino acid sequence of SEQ ID NO: 59; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 52; VL2 contains the amino acid sequence of SEQ ID NO: 60; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 45; VL2 contains the amino acid sequence of SEQ ID NO: 53; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 46; V L2 The amino acid sequence containing SEQ ID NO:54; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 47; V L2 Contains the amino acid sequence of SEQ ID NO: 55; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 48; V L2 Contains the amino acid sequence of SEQ ID NO: 56; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 49; V L2 Contains the amino acid sequence of SEQ ID NO: 57; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 50; V L2 Contains the amino acid sequence of SEQ ID NO: 58; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO:51; V L2 Contains the amino acid sequence of SEQ ID NO: 59; or V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 52; V L2 It contains the amino acid sequence of SEQ ID NO: 60.

[0149] In some embodiments, polypeptide (I) consists of the amino acid sequence of SEQ ID NO: 64; polypeptide (II) consists of the amino acid sequence of SEQ ID NO: 65; and polypeptide (III) consists of the amino acid sequence of SEQ ID NO: 66.

[0150] In one aspect, this article provides a method for treating or preventing leukemia in a pediatric subject in need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46.

[0151] In some embodiments, the leukemia is acute myeloid leukemia (AML). In some embodiments, the AML is relapsed or refractory. In some embodiments, the leukemia is B-cell acute lymphoblastic leukemia (B-ALL).

[0152] In some embodiments, the binding protein is administered to the subject intravenously, subcutaneously, intraperitoneally, or intramuscularly. In some embodiments, the binding protein is administered to the subject intravenously.

[0153] In some embodiments, the first antigen-binding domain having binding specificity to CD123 includes:

[0154] The heavy chain variable domain (VH1) comprises CDR-H1, H2, and H3 corresponding to amino acid sequences of SEQ ID NO: 1, 2, and 3, or corresponding to amino acid sequences of SEQ ID NO: 4, 5, and 5, respectively; and

[0155] The light chain variable domain (VL1) contains CDR-L1, L2 and L3 corresponding to amino acid sequences of SEQ ID NO: 7, 8 and 9, or amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively.

[0156] In some embodiments, VH1 contains at least about 90% of the amino acid sequence identical to that of SEQ ID NO: 41, and wherein VL1 contains at least about 90% of the amino acid sequence identical to that of SEQ ID NO: 43; or VH1 contains at least about 90% of the amino acid sequence identical to that of SEQ ID NO: 42, and wherein VL1 contains at least about 90% of the amino acid sequence identical to that of SEQ ID NO: 44.

[0157] In some embodiments, the VH1 comprises the amino acid sequence of SEQ ID NO: 41, and the VL1 comprises the amino acid sequence of SEQ ID NO: 43; or the VH1 comprises the amino acid sequence of SEQ ID NO: 42, and the VL1 comprises the amino acid sequence of SEQ ID NO: 44.

[0158] In some embodiments, the second antigen-binding domain having binding specificity to NKp46 comprises: a second heavy chain variable domain (VH2) comprising CDRs-H1, H2, and H3 corresponding to the following amino acid sequences: respectively, SEQ ID NO: 13, 14, and 15; respectively, SEQ ID NO: 16, 17, and 18; respectively, SEQ ID NO: 19, 20, and 21; respectively, SEQ ID NO: 22, 23, and 24; or respectively, SEQ ID NO: 16, 25, and 26; and a second light chain variable domain (VL2) comprising CDRs-L1, L2, and L3 corresponding to the following amino acid sequences: respectively, SEQ ID NO: 27, 28, and 29; respectively, SEQ ID NO: 30, 31, and 32; respectively, SEQ ID NO: 33, 34, and 35; respectively, SEQ ID NO: 36, 37, and 38; or respectively, SEQ ID NO: 39, 31 and 40.

[0159] In some embodiments, VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 45, and wherein VL1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 53; VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 46, and wherein VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 54; VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 47, and wherein VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 55; VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 48, and wherein VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 56; VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and wherein VL ... The amino acid sequence of SEQ ID NO: 57 is at least about 90% identical to the amino acid sequence of SEQ ID NO: 50; the VH2 contains at least about 90% identical to the amino acid sequence of SEQ ID NO: 58; the VH2 contains at least about 90% identical to the amino acid sequence of SEQ ID NO: 51; the VL2 contains at least about 90% identical to the amino acid sequence of SEQ ID NO: 59; or the VH2 contains at least about 90% identical to the amino acid sequence of SEQ ID NO: 52; the VL2 contains at least about 90% identical to the amino acid sequence of SEQ ID NO: 60.

[0160] In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 45, and wherein VL1 comprises the amino acid sequence of SEQ ID NO: 53; VH2 comprises the amino acid sequence of SEQ ID NO: 46, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 54; VH2 comprises the amino acid sequence of SEQ ID NO: 47, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 55; VH2 comprises the amino acid sequence of SEQ ID NO: 48, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 56; VH2 comprises the amino acid sequence of SEQ ID NO: 49, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 57; VH2 comprises the amino acid sequence of SEQ ID NO: 50, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 58; VH2 comprises the amino acid sequence of SEQ ID NO: 51, and wherein VL2 comprises the amino acid sequence of SEQ ID NO: 59; or VH2 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of SEQ ID NO: 52, wherein the VL2 comprises the amino acid sequence of SEQ ID NO: 60.

[0161] In some embodiments, the binding protein comprises three polypeptide chains (I), (II), and (III) that form two ABDs, as defined below:

[0162] V 1A - C 1A - Hinge 1 - (C H 2-C H 3) A (I)

[0163] V 1B - C 1B - Hinge 2 - (C H 2-C H 3) B - L1 - V 2A - C 2A - Hinge 3(II)

[0164] V 2B - C 2B (III)

[0165] in:

[0166] V 1A and V 1B Forming binding pairs V1 (V H1 / VL1 );

[0167] V 2A and V 2B Forming binding pairs V2 (V H2 / V L2 );

[0168] C 1A and C 1B Forming a pair of C1 (C H 1 / C L ), and C 2A and C 2B Formation of C2 (C H 1 / C L ), where C H 1 represents the immunoglobulin heavy chain constant domain 1, and C L It is the constant structural domain of the immunoglobulin light chain;

[0169] Hinge 1, hinge 2 and hinge 3 may be the same or different, and correspond to all or part of the immunoglobulin hinge region;

[0170] (C H 2-C H 3) A and (C) H 2-C H 3) B For the same or different, and containing the immunoglobulin heavy chain constant domain 2 (C H 2) and immunoglobulin heavy chain constant domain 3 (C H 3);

[0171] L1 is an amino acid linker.

[0172] In some implementation schemes, C 1B For immunoglobulin heavy chain constant domain 1 (C H 1);

[0173] C 2A For immunoglobulin heavy chain constant domain 1 (C H 1);

[0174] C L Corresponding to the immunoglobulin κ light chain constant domain (C κ );

[0175] (C H 2-C H 3) A The amino acid sequence corresponding to SEQ ID NO: 69;

[0176] (C H 2-CH 3) B The amino acid sequence corresponding to SEQ ID NO: 70;

[0177] Hinge 1 corresponds to the amino acid sequence of SEQ ID NO: 74;

[0178] Hinge 2 corresponds to the amino acid sequence of SEQ ID NO: 75;

[0179] Hinge 3 corresponds to the amino acid sequence of SEQ ID NO: 77;

[0180] L1 corresponds to the amino acid sequence of SEQ ID NO: 76.

[0181] In some embodiments, residue N297 of the Fc region or a variant thereof, according to EU number, contains an N-linked glycosylation. In some embodiments, all or part of the Fc region or a variant thereof binds to the human CD16A (FcγRIII) polypeptide. In some embodiments, the binding protein comprises at least two polypeptide chains linked by at least one disulfide bond.

[0182] In some embodiments, the polypeptide chain (I) and the polypeptide chain (II) are coupled via C 1A At least one disulfide bond is connected between hinge 2 and the polypeptide chain (II) and the polypeptide chain (III) via hinge 3 to C. 2B At least one disulfide bond connects them.

[0183] In some implementation schemes, V 1A For V L1 And V 1B For V H1 In some implementations, V 2A For V H2 And V 2B For V L2 .

[0184] In some implementation schemes, V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2The CDR-H1 contains the amino acid sequence of SEQ ID NO: 13; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 14; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 15; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 27; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 28; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 29; V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 17; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 18; V L2 CDR-L1 containing the amino acid sequence of SEQ ID NO: 30; CDR-L2 containing the amino acid sequence of SEQ ID NO: 31; CDR-L3 containing the amino acid sequence of SEQ ID NO: 32; V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 19; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 20; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 21; V L2 CDR-L1 containing the amino acid sequence of SEQ ID NO: 33; CDR-L2 containing the amino acid sequence of SEQ ID NO: 34; CDR-L3 containing the amino acid sequence of SEQ ID NO: 35; VH1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 22; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 23; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 24; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 36; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 37; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 38; V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 25; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 26; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 39; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 40; V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 13; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 14; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 15; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 27; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 28; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 29; V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 17; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 18; V L2 CDR-L1 containing the amino acid sequence of SEQ ID NO: 30; CDR-L2 containing the amino acid sequence of SEQ ID NO: 31; CDR-L3 containing the amino acid sequence of SEQ ID NO: 32; V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2The CDR-H1 contains the amino acid sequence of SEQ ID NO: 19; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 20; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 21; V L2 CDR-L1 containing the amino acid sequence of SEQ ID NO: 33; CDR-L2 containing the amino acid sequence of SEQ ID NO: 34; CDR-L3 containing the amino acid sequence of SEQ ID NO: 35; V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 22; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 23; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 24; V L2 The CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; the CDR-L2 containing the amino acid sequence of SEQ ID NO: 37; the CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; or V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 25; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 26; V L2The CDR-L1 contains the amino acid sequence of SEQ ID NO: 39; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 40.

[0185] In some implementation schemes, V H1 and V L1 The amino acid sequences corresponding to SEQ ID NO: 41 and 43, or the amino acid sequences corresponding to SEQ ID NO: 42 and 44, respectively; and / or V H2 and V L2 Corresponding to

[0186] The amino acid sequences of SEQ ID NO: 45 and 53;

[0187] The amino acid sequences of SEQ ID NO: 46 and 54;

[0188] The amino acid sequences of SEQ ID NO: 47 and 55;

[0189] The amino acid sequences of SEQ ID NO: 48 and 56;

[0190] The amino acid sequences of SEQ ID NO: 49 and 57;

[0191] The amino acid sequences of SEQ ID NO: 50 and 58;

[0192] The amino acid sequences of SEQ ID NO: 51 and 59; or

[0193] The amino acid sequences of SEQ ID NO: 52 and 60.

[0194] In some implementation schemes, V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 45; V L2 Contains the amino acid sequence of SEQ ID NO: 53; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 46; V L2 Contains the amino acid sequence of SEQ ID NO: 54; V H1 Contains the amino acid sequence of SEQ ID NO: 41; VL1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 47; V L2 Contains the amino acid sequence of SEQ ID NO: 55; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 48; V L2 Contains the amino acid sequence of SEQ ID NO: 56; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 49; VL2 contains the amino acid sequence of SEQ ID NO: 57; V H1 Contains the amino acid sequence of SEQ ID NO:41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 50; V L2 Contains the amino acid sequence of SEQ ID NO: 58; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 51; V L2 Contains the amino acid sequence of SEQ ID NO: 59; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 52; VL2 contains the amino acid sequence of SEQ ID NO: 60; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 45; VL2 contains the amino acid sequence of SEQ ID NO: 53; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 46; V L2 The amino acid sequence containing SEQ ID NO:54; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 47; V L2 Contains the amino acid sequence of SEQ ID NO: 55; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 48; V L2 Contains the amino acid sequence of SEQ ID NO: 56; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 49; VL2 contains the amino acid sequence of SEQ ID NO: 57; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 50; V L2 Contains the amino acid sequence of SEQ ID NO: 58; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO:51; V L2 Contains the amino acid sequence of SEQ ID NO: 59; V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 52; VL2 contains the amino acid sequence of SEQ ID NO: 60; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1Contains the amino acid sequence of SEQ ID NO: 44; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 45; VL2 contains the amino acid sequence of SEQ ID NO: 53; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 46; V L2 Contains the amino acid sequence of SEQ ID NO: 54; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 47; V L2 Contains the amino acid sequence of SEQ ID NO: 55; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 The amino acid sequence containing SEQ ID NO:44; V H2 Contains the amino acid sequence of SEQ ID NO: 48; V L2 Contains the amino acid sequence of SEQ ID NO: 56; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 49; V L2 Contains the amino acid sequence of SEQ ID NO: 57; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 50; V L2 Contains the amino acid sequence of SEQ ID NO: 58; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 51; V L2 Contains the amino acid sequence of SEQ ID NO: 59; V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 52; V L2 It contains the amino acid sequence of SEQ ID NO: 60.

[0195] In some embodiments, polypeptide (I) consists of the amino acid sequence of SEQ ID NO: 64; polypeptide (II) consists of the amino acid sequence of SEQ ID NO: 65; and polypeptide (III) consists of the amino acid sequence of SEQ ID NO: 66.

[0196] In one aspect, this article provides a method for treating or preventing blastic plasmacytoid dendritic cell tumors (BPDCN) in a subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46.

[0197] The first antigen-binding domain comprises a heavy chain variable domain (VH1) and a light chain variable domain (VL1), wherein:

[0198] The VH1 contains complementarity-determining regions (CDRs) - H1, H2, and H3 corresponding to the amino acid sequences of SEQ ID NO: 1, 2, and 3; and

[0199] The VL1 comprises CDR-L1, L2, and L3 corresponding to the amino acid sequences of SEQ ID NO: 7, 8, and 9; and

[0200] The second antigen-binding domain comprises a heavy chain variable domain (VH2) and a light chain variable domain (VL2), wherein:

[0201] The VH2 contains CDR-H1, H2, and H3 corresponding to the amino acid sequences of SEQ ID NO: 13, 14, and 15; and

[0202] The VL2 comprises CDR-L1, L2, and L3 corresponding to the amino acid sequences of SEQ ID NO: 27, 28, and 29;

[0203] Furthermore, all or part of the Fc region of the immunoglobulin or its variants bind to the human Fc-γ receptor.

[0204] On the other hand, this article provides a method for treating or preventing blastic plasmacytoid dendritic cell tumors (BPDCN) in a pediatric subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123, a second antigen-binding domain having binding specificity to NKp46, and all or part of an immunoglobulin Fc region or a variant thereof that binds to a human Fc-γ receptor, wherein the binding protein comprises:

[0205] A polypeptide (I) consisting of the amino acid sequence of SEQ ID NO: 64;

[0206] A polypeptide (II) consisting of the amino acid sequence of SEQ ID NO: 65; and

[0207] A polypeptide (III) consisting of the amino acid sequence of SEQ ID NO: 66.

[0208] On the other hand, this article provides a method for treating or preventing blastic plasmacytoid dendritic cell tumors (BPDCN) in pediatric subjects of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46.

[0209] The first antigen-binding domain comprises a heavy chain variable domain (VH1) and a light chain variable domain (VL1), wherein:

[0210] The VH1 contains complementarity-determining regions (CDRs) - H1, H2, and H3 corresponding to the amino acid sequences of SEQ ID NO: 1, 2, and 3; and

[0211] The VL1 comprises CDR-L1, L2, and L3 corresponding to the amino acid sequences of SEQ ID NO: 7, 8, and 9; and

[0212] The second antigen-binding domain comprises a heavy chain variable domain (VH2) and a light chain variable domain (VL2), wherein:

[0213] The VH2 contains CDR-H1, H2, and H3 corresponding to the amino acid sequences of SEQ ID NO: 13, 14, and 15; and

[0214] The VL2 comprises CDR-L1, L2, and L3 corresponding to the amino acid sequences of SEQ ID NO: 27, 28, and 29;

[0215] Furthermore, all or part of the Fc region of the immunoglobulin or its variants bind to the human Fc-γ receptor.

[0216] On the other hand, this article provides a method for treating or preventing blastic plasmacytoid dendritic cell tumor (BPDCN) in a subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123, a second antigen-binding domain having binding specificity to NKp46, and all or part of an immunoglobulin Fc region or a variant thereof that binds to a human Fc-γ receptor, wherein the binding protein comprises:

[0217] A polypeptide (I) consisting of the amino acid sequence of SEQ ID NO: 64;

[0218] A polypeptide (II) consisting of the amino acid sequence of SEQ ID NO: 65; and

[0219] A polypeptide (III) consisting of the amino acid sequence of SEQ ID NO: 66.

[0220] In one aspect, this article provides a method for treating or preventing acute myeloid leukemia (AML) in a pediatric subject in need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46.

[0221] In some implementations, the pediatric subjects with AML are between approximately 1 and approximately 11 years old.

[0222] In one aspect, this article provides a method for treating or preventing acute myeloid leukemia (AML) in a pediatric subject in need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46, wherein the pediatric subject is between about 1 year and about 11 years old.

[0223] On the other hand, this article provides a method for treating or preventing acute myeloid leukemia (AML) in a pediatric subject in need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46, wherein the first antigen-binding domain comprises a heavy chain variable domain (VH1) and a light chain variable domain (VL1), wherein: the VH1 comprises complementarity-determining regions (CDRs) corresponding to the amino acid sequences of SEQ ID NO: 1, 2, and 3 - H1, H2, and H3; and the VL1 comprises CDRs corresponding to the amino acid sequences of SEQ ID NO: 7, 8, and 9 - L1, L2, and L3; and wherein the second antigen-binding domain comprises a heavy chain variable domain (VH2) and a light chain variable domain (VL2), wherein: the VH2 comprises CDRs corresponding to the amino acid sequences of SEQ ID NO: 13, 14, and 15 - H1, H2, and H3; and the VL2 comprises CDRs corresponding to the amino acid sequences of SEQ ID NO: 13, 14, and 15 - L1, H2, and L3; and the VL2 comprises CDRs corresponding to the amino acid sequences of SEQ ID NO: 13, 14, and 15 - L1, L ... The amino acid sequences 27, 28, and 29 are CDR-L1, L2, and L3; and wherein all or part of the immunoglobulin Fc region or a variant thereof binds to the human Fc-γ receptor, and wherein the age of the pediatric subject in need is between approximately 1 year and approximately 11 years.

[0224] On the other hand, this article provides a method for treating or preventing acute myeloid leukemia (AML) in a pediatric subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123, a second antigen-binding domain having binding specificity to NKp46, and all or part of an immunoglobulin Fc region or a variant thereof that binds to a human Fc-γ receptor, wherein the binding protein comprises: a polypeptide (I) consisting of the amino acid sequence of SEQ ID NO: 64; a polypeptide (II) consisting of the amino acid sequence of SEQ ID NO: 65; and a polypeptide (III) consisting of the amino acid sequence of SEQ ID NO: 66, and wherein the pediatric subject of need is between about 1 year and about 11 years old.

[0225] In some embodiments, the pediatric subject is between about 1 year and about 17 years old. In some embodiments, the pediatric subject is between about 1 year and about 11 years old. In some embodiments, the pediatric subject is about 1 year old. In some embodiments, the pediatric subject is about 2 years old. In some embodiments, the pediatric subject is about 3 years old. In some embodiments, the pediatric subject is about 4 years old. In some embodiments, the pediatric subject is about 5 years old. In some embodiments, the pediatric subject is about 4 years old. In some embodiments, the pediatric subject is about 6 years old. In some embodiments, the pediatric subject is about 7 years old. In some embodiments, the pediatric subject is about 8 years old. In some embodiments, the pediatric subject is about 9 years old. In some embodiments, the pediatric subject is about 10 years old. In some embodiments, the pediatric subject is about 11 years old. In some embodiments, the pediatric subject is about 12 years old. In some embodiments, the pediatric subject is about 13 years old. In some embodiments, the pediatric subject is about 14 years old. In some embodiments, the pediatric subject is approximately 15 years old. In some embodiments, the pediatric subject is approximately 16 years old. In some embodiments, the pediatric subject is approximately 17 years old. Attached Figure Description

[0226] Unless otherwise stated, in accordance with standard usage and convention, the binding protein of this disclosure is oriented at the amino-terminal (“N-term” or “N-term”) on the left-hand side and at the carboxyl-terminal (“C-term” or “C-term”) on the right-hand side.

[0227] Figure 1 depicts a three-dimensional schematic representation of the F25 form, a variant of the bispecific F5 form, including a human NKp46 binding site and a human CD123 binding site. As depicted in Figure 1, the C-terminus of the peptide is on the left-handed side, and the N-terminus is on the right-handed side.

[0228] Figures 2A-2D show two-dimensional schematic representations of the F25, F5, F26, and F6 forms, respectively, including the relevant domains of each polypeptide chain. In Figures 2A-2D, the C-terminus of the polypeptide is on the left-hand side, and the N-terminus is on the right-hand side. The human NKp46 binding domain is formed by the VH / VL pair on the left. The human CD123 binding domain is formed by the VH / VL pair on the right.

[0229] Figure 2A shows a two-dimensional schematic representation of F25. This representation represents the claimed “NKp46-CD123_F25” binding protein.

[0230] Figure 2B shows a two-dimensional schematic representation of the F5 form. The difference between F5 and F25 is that the CL and CH pairs of the NKp46 binding domain are swapped, and the third polypeptide chain contains both a CH1 domain and a VL domain.

[0231] Figure 2C shows a two-dimensional schematic representation of the F26 form. This F26 differs from F25 in Figure 2A in that it includes an Fc-silencing N297S mutation in each CH2 domain.

[0232] Figure 2D shows a two-dimensional schematic representation of the F6 form. This F6 differs from F5 in Figure 2B in that it includes an Fc-silencing N297S mutation in each CH2 domain.

[0233] Figure 2E shows a two-dimensional detailed representation of the variant of the F25 form. This F25 form representation corresponds to the F25 form in Figure 2A.

[0234] Figure 3 illustrates the proposed mechanism of action of the NK cell engager (NKCE) in killing tumor cells expressing CD123 (i.e., the AML cell line; MOLM-13) and NK cells expressing NKp46 and Fcγ receptor (CD16a) after combined binding. Reprinted and adapted from Gauthier, L. et al. (“Multifunctional natural killer cellengagers targeting NKp46 trigger protective tumor immunity”. Cell 177, 1701-1713 (2019)).

[0235] Figures 4A-4C are a set of schematic diagrams showing the in vitro model of synapse-driven PK / PD in MOLM-13 cells (Figure 4A), the in vivo NHP model of synapse-driven PK / PD in vivo (Figure 4B), and the synapse-driven PK / PD model of transformed AML (Figure 4C).

[0236] Figure 5 depicts the cytotoxicity (%) curves of different CD123 NKCE concentrations in the in vitro system, stratified by incubation time and E:T ratio in the MOLM-13 in vitro model. Solid lines represent typical model simulations; solid circles represent individual observations of in vitro cytotoxicity.

[0237] Figures 6A-6C show visual predictions of PK (Figure 6A), total CD123+ cells (Figure 6B), and NKp46+ NK cells (Figure 6C) in an in vivo NHP PK / PD model for intravenous administration of 3000 µg / kg SD, 100 and 3000 µg / kg QW, and subcutaneous administration of 3.6 µg / kg SD. PK data are also included from intravenous administration of 100 µg / kg and 3000 µg / kg biw, and subcutaneous administration of 3000 µg / kg biw.

[0238] Figures 7A-7B show the predicted CD123 NKCE after intra-patient dose escalation regimens using the biw regimen (where 10 µg / kg and 30 µg / kg were the first and second doses in week 1, followed by 100 µg / kg (10 / 30 / 100)) and a fixed-dose biw regimen of 100 µg / kg (fixed 100). Figure 7A shows the PK prediction results on a semi-logarithmic time-course plot. The horizontal dashed line represents the minimum concentration (1.7 nmol / L) showing mild cytokine release in whole blood cytokine release assays. Figure 7B shows the CFB% in the predicted CD123+ cell count. The horizontal dashed line represents changes of 0 and -95% relative to baseline (target levels for efficacy definition).

[0239] Figure 8 shows the individual model fit of the NHP PK / PD model from representative individuals. Dots represent observations, and lines represent individual predictions.

[0240] Figure 9 shows a set of graphs illustrating the external validation of in vivo NHP PK / PD model simulations by comparing observed total CD123+ and NKp46+ NK cell counts after biw administration with the PK data from this dosing regimen to estimate PK parameters. Solid lines represent typical model simulations, and shaded areas represent the 90% prediction interval (N=100). Solid circles represent the mean of the observed results, with standard deviations used as error bars. PK parameters were estimated using observed PK data (red solid circles) while comparing observed total CD123+ and NKp46+ NK cell counts (blue solid circles) with the model simulation curves. Note: CD123+ cell counts > 200 cells / µL were excluded.

[0241] Figures 10A-10B show the Global Sensitivity Analysis (GSA) and Local Sensitivity Analysis (LSA). Figure 10A uses the Extended Fourier Amplitude Sensitivity Test (eFAST) to evaluate the predicted AUC parameter (k) of CD123+ cells at the selected FIH starting dose (10 / 30 / 100 µg / kg) in the transformed AML PK / PD model. g alpha [α], k in TP, gamma [γ], k max Beta [β], k int EC 50 TC max ,RTdE,EF0,TA0,k out Slp NK ,RTdT,KD1,KD2,k off1 and k off2 Sensitivity of CD123+ cell counts at the initial FIH dose. Figure 10B (LSA) evaluates the percentage change in CD123+ cell counts relative to baseline in GSA as a sensitive parameter identified.

[0242] Figure 11 shows the Kaplan-Mail curves of mice treated with CD123 NKCE and anti-CD123 antibody-drug conjugates and the differences in survival rates between them.

[0243] Figure 12 depicts the Kaplan-Mail curves of NK-depleted SCID mice treated with muNKp46-huCD123, X-huCD123 isotype control, and muNKp46-X isotype control.

[0244] Figures 13A-13C illustrate the outcomes of responders at different time points after treatment. The upper figure depicts patients who received CD123 NKCEs with DL3 and DL4. Both responders maintained remission (CR) at 8.8 months and 12.2 months after treatment. The lower figure depicts patients at other dose levels.

[0245] Figures 14A, 14B, 14C, and 14D depict the leukemia blast cell counts in whole blood (Figures 14A and 14D) and bone marrow (Figures 14B and 14C) before and after treatment with CD123 NKCE.

[0246] Figures 15A and 15B illustrate the CD123 NKCE levels of DL1-DL6 in patients throughout cycle 1. 峰 (Figure 15A) and C 谷 (Figure 15B) (Measured in ng / mL).

[0247] Figures 16A and 16B show the mean plasma concentrations at dose levels DL1–DL6 during cycle 1. Linearity was achieved at a weekly dose of 3000 µg / kg (DL5 and DL6).

[0248] Figure 17 shows plasma cytokine levels at dose levels DL1–DL6 during cycle 1. Key pro-inflammatory cytokine levels peaked between 1 and 3 hours after the first dose and returned to baseline by 24 hours.

[0249] Figures 18A-18C depict escalating treatment protocols (Figure 18A) and extended treatment protocols (Figure 18B) for adults. Figure 18C depicts escalating / extended / optimized treatment protocols for pediatrics.

[0250] Figure 19 illustrates the study design for each cohort (cohorts A, B, C, and D) in both the adult study (top) and the pediatric study (bottom) in the dose escalation and expansion / optimization sections.

[0251] Figure 20 depicts the cytotoxicity (lysis %) of MOLM-12, THP-1, OCI-AML2, and NB-4 cells incubated with a series of CD123 NKCE or NKp46xCD16-X isotype control concentrations. Figure 21 shows a set of simulated CD123 NKCE plasma concentration versus time curves, overlaid with observational data from the first induction cycle.

[0252] Figure 22 shows a set of simulated synaptic concentration versus time curves during the first induction cycle.

[0253] Figure 23 shows the standardized synaptic exposure predicted by dose level for individuals during the first induction cycle (n = 58 patients).

[0254] Figure 24 shows the predicted response rates (CR / CRi or BR) based on dose levels.

[0255] Figure 25 shows the predicted synaptic exposure based on dose level. Detailed Implementation

[0256] This disclosure provides a method for treating hematologic disorders and conditions, such as relapsed or refractory acute myeloid leukemia (AML), B-cell acute lymphoblastic lymphoma (B-ALL), high-risk myelodyplastic or blastic plasmacytoid dendritic cell tumors (BPDCN), the method comprising a multifunctional binding protein that binds to a surface biomarker (i.e., NKp46) on immune NK cells and a target antigen (i.e., CD123) on tumor target cells, and is capable of repositioning NK cells to lyse target cells expressing the CD123 surface biomarker. The multifunctional binding protein of this disclosure further comprises all or part of an Fc region or a variant thereof, which binds all or part of an Fc-γ receptor (FcγR), particularly activating an Fc-γ receptor (FcγR), such as FcγRIIIa (also known as CD16a).

[0257] I. Definition

[0258] To facilitate understanding of this disclosure, selected terms are defined below.

[0259] The articles “a” and “an” are used in this text to refer to one or more grammatical objects of the article (i.e., at least one or more).

[0260] As used herein, when referring to measurable values ​​(such as quantities, time intervals, etc.), “about” or “approximately” is intended to cover variations of ±20% or ±10%, ±5%, ±1% or ±0.1% of a given value or range, as such variations are appropriate for the methods disclosed.

[0261] As used herein, the term “differentiation cluster 123” or “CD123” marker is also referred to as “interleukin-3 receptor α (IL3RA)” or “IL3R,” “IL3RX,” “IL3RY,” “IL3RAY,” or “hIL-3Ra,” and represents the interleukin-3 specific subunit of the heterodimeric cytokine receptor. The functional interleukin-3 receptor is a heterodimer containing a specific α chain (IL-3A; CD123) shared with the receptors for granulocyte-macrophage community-stimulating factor (GM-CSF) and interleukin-5 (IL-5) and an IL-3 receptor β chain (βθ; CD131). CD123 is a type I integrated transmembrane protein with an estimated molecular weight of approximately 43 kDa, containing an extracellular domain involved in IL-3 binding, a transmembrane domain, and a short cytoplasmic tail of approximately 50 amino acids. The extracellular domain consists of two regions: an N-terminal region of approximately 100 amino acids, whose sequence shows similarity to the equivalent regions of the GM-CSF and IL-5 receptor α chains; and a region adjacent to the transmembrane domain containing four conserved cysteine ​​residues and a motif shared with other members of this cytokine receptor family. The IL-3 binding domain contains a cytokine receptor motif (CRM) of approximately 200 amino acid residues, consisting of two Ig-like folded domains. The extracellular domain of CD123 is highly glycosylated, with N-glycosylation essential for both ligand binding and receptor signaling. The protein family comprises three members: IL3RA (CD123A), CSF2RA, and IL5RA. The overall structure of these three members is highly conserved, but sequence homology is very low. To date, a 300-amino acid isoform of CD123 has been identified, but only at the RNA level; it is available in the Getentry database with accession number ACM241 16.1. The reference sequence of the full-length human CD123 protein (including the signaling peptide) is available from the NCBI database with accession number NP_002174.1 and Uniprot accession number P26951.

[0262] The extracellular domain (ECD) of human CD123 consists of the amino acid sequence of SEQ ID NO: 82. CD123 (interleukin-3 receptor α chain IL-3Ra) is a tumor antigen overexpressed in a variety of hematologic malignancies. Most AML blast cells express surface CD123, and this expression does not vary with AML subtype. Higher expression of CD123 on AML at diagnosis has been reported to be associated with a poorer prognosis.

[0263] CD123 expression has been reported in other hematologic malignancies, including myelodysplastic syndrome, systemic obesity cell neoplasm, blastic plasmacytoid dendritic cell neoplasm (BPDCN), ALL, and hairy cell leukemia.

[0264] As used herein, “natural killer” or “NK cells” refers to a subset of lymphocytes involved in unconventional immunity. NK cells can be identified by certain characteristics and biological properties, such as the expression of specific surface antigens (including CD16, CD56, and / or CD57, and NKp46 for human NK cells); the absence of α / β or γ / δ TCR complexes on the cell surface; the ability to bind to and kill cells that do not express “self” MHC / HLA antigens by activating specific cytolysis machinery; the ability to kill tumor cells or other diseased cells expressing ligands of NK activation receptors; and the ability to release protein molecules that stimulate or suppress immune responses (called cytokines). Any of these characteristics and activities can be used to identify NK cells using methods well known in the art. The term NK cell will also cover any subset of NK cells. In the context of this document, “active” NK cells refer to biologically active NK cells, including NK cells with the ability to lyse target cells or enhance the immune function of other cells. NK cells can be obtained using various techniques known in the art, such as isolation from blood samples, cytapheresis, tissue or cell collection, etc. Useful protocols for assays involving NK cells can be found in Natural Killer Cells Protocols (edited by Campbell KS and Colonna M). HumanPress. pp. 219-238 (2000).

[0265] As used herein, the term “NKp46” marker or “natural cytotoxicity trigger receptor 1” (also known as “CD335”, “NKP46”, “NK-p46”, or “LY94”) refers to the protein or polypeptide encoded by the Ncr1 gene. A reference sequence of the full-length human NKp46 protein is available from the NCBI database under accession number NP_004820. The extracellular domain (ECD) of human NKp46 corresponds to the amino acid sequence of SEQ ID NO: 80. The human NKp46 mRNA sequence is described under NCBI accession number NM_004829.

[0266] As used herein, the terms “Fc-γ receptor” or “FcγR” or “Fc-gamma receptor” can refer to both activating and inhibitory FcγRs. Fc-γ receptors (FcγRs) are cellular receptors for the Fc region of immunoglobulin G (IgG). Upon binding to complex IgG, FcγRs can modulate the function of cellular immune effectors, thereby connecting the adaptive and innate immune systems, including ADCC-mediated immune responses. In humans, six classic FcγRs have been reported: one high-affinity receptor (FcγRI) and five low- to medium-affinity FcγRs (FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, and FcγRIIIB). All FcγRs bind to the same region on the IgG Fc region, but with different high (FcgRI) and low (FcgRII and FcgRIII) affinities. At the functional level, most FcγRs are activating receptors that can induce the cellular responses mentioned above, including ADCC-mediated immune responses. However, FcγRI, FcγRIIa, FcγRIIc, and FcγRIIIa are activating receptors characterized by the intracellular immune receptor tyrosine activation motif (ITAM), and FcγRIIb has an inhibitory motif (ITIM) and is therefore inhibitory. Unless otherwise stated, the term FcγR encompasses activating receptors including FcγRI (CD64), FcγRIIA (CD32a), FcγRIIIa (CD16a), and FcγRIIIb (CD16b), with FcγRIIIa (CD16a) being preferred.

[0267] As used herein, the terms “FcγRIIIa (CD16a)”, “FcγRIIIa”, “CD16a”, “CD16”, or “differentiation cluster 16” can refer to a 50-65 kDa cell surface molecule expressed as a transmembrane receptor on obese cells, macrophages, and natural killer cells. FcγRIIIa is an activated receptor containing an immunoreceptor tyrosine activation motif (ITAM) in the associated FcRγ chain; ITAM is essential for receptor expression, surface assembly, and signaling. CD16a is a low-affinity receptor for IgG and an important receptor for NK cell-mediated ADCC (antibody-dependent cell-mediated cytotoxicity). The high-affinity receptor CD16a is preferentially present on NK cells and monocytes and induces antibody-dependent cytotoxicity (ADCC) upon IgG binding.

[0268] As used herein, the terms “form 5” or “F5”, “form 25” or “F25”, “form F6” or “F6”, and “form 26” or “F26” refer to specific binding protein conformations of bispecific or multispecific antibodies specifically engineered to incorporate multiple antigen-binding domains into a single antibody molecule. The present disclosure discloses a multifunctional binding protein comprising an NKp46 binding domain and a CD123 binding domain, prepared based on the F25 form. F25 and form F26 differ from forms F5 and F6 in that a CH1 / CL pair between the second and third polypeptide chains is swapped to form a CL / CH1 pair. Forms F5 and F6 have previously been described in International Patent Application WO 2017114694 (incorporated herein by reference).

[0269] As used herein, the term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets (e.g., human NKp46 and human CD123) through at least two different antigen-binding domains (ABDs). Bispecific binding proteins can be bivalent (two ABDs) or multivalent (more than two ABDs).

[0270] As used herein, the terms “specifically binds to” or “has binding specificity to” refer to an antigen-binding domain (ABD) with a binding radius of at least about 1 x 10⁻⁶. -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, lx 10 -12 The ability of M or more Kd to bind to antigens containing epitopes (e.g., human NKp46 and / or human CD123), and / or the ability to bind to epitopes with an affinity at least twice that of the antigen-binding domain for nonspecific antigens.

[0271] As used herein, the term “specific binding to human NKp46 peptide” may refer to specific binding to a peptide containing the amino acid sequence of SEQ ID NO: 80.

[0272] As used herein, the term “specific binding to human CD123 peptide” may refer to specific binding to a peptide containing the amino acid sequence of SEQ ID NO: 82.

[0273] As used herein, the term “binding to human Fc-γ receptor peptide” may refer to the binding of a peptide containing the amino acid sequence of SEQ ID NO: 83 or SEQ ID NO: 84.

[0274] Competitive binding assays and other methods for determining specific binding are further described below and are well known in the art. Expressions such as “specifically binds to” or “has binding specificity for” are used interchangeably. The terms are not to be construed as exclusively referring to those antibodies, peptides, and / or multi-chain peptides that actually bind to the target / binding partner, but also to those antibodies, peptides, and / or multi-chain peptides that retain specificity for the target, even though provided in an unbound form. Binding specificity can be determined by the affinity constant KA (or K0). A ) and dissociation constant KD (or K D ) Quantitatively determine.

[0275] As used in this article, the terms "affinity" and "concentration" (EC) are used interchangeably. 50 ) or equilibrium dissociation constant (K D The affinity of an antibody or peptide to an epitope is indicated by the strength of that binding. The affinity of an antibody is given by a specific type of equilibrium constant, namely the dissociation constant K. DThe affinity constant KA is defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant KA is defined by 1 / KD. Preferred methods for determining the affinity of mAbs can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988); Coligan et al. (eds.), Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993); and Muller, Meth. Enzymol. 92:589-601 (1983), all of which are incorporated herein by reference in their entirety. A preferred standard method well known in the art for determining the affinity of mAbs is screening using surface plasma resonance (SPR) (e.g., by analysis using a BIAcore™ SPR analyzer). KD less than 50 nM, as determined in a non-restrictive manner, such as by SPR under physiological conditions (e.g., under normal buffer conditions at pH range 6 to 8), can generally be considered as an indicator of binding specificity of antigen-antigen binding domain (ABD) interactions.

[0276] As an illustration, and according to some specific exemplary embodiments, the binding proteins reported herein include:

[0277] - Under physiological conditions, as measured by SPR, with K < 10 nM D Especially with K less than 0.5 nM D An antigen-binding domain that specifically binds to human CD123;

[0278] - Under physiological conditions, as measured by SPR, with K < 50 nM D Especially with K less than 20 nM D An antigen-binding domain that specifically binds to human NKp46.

[0279] As used herein, the term “and / or” is a grammatical conjunction that will be interpreted to cover one or more possible scenarios that it connects to. For example, the phrase “such natural sequence proteins can be prepared using standard recombinant and / or synthetic methods” indicates that the natural sequence protein can be prepared using standard recombinant and synthetic methods, or that the natural sequence protein can be prepared using standard recombinant methods, or that the natural sequence protein can be prepared using synthetic methods.

[0280] As used herein, “treatment” refers to therapeutic use (i.e., to a subject suffering from a given disease) and means reversing one or more symptoms of such a disorder or condition, alleviating one or more symptoms of such a disorder or condition, or inhibiting the progression of one or more symptoms of such a disorder or condition. Therefore, treatment refers not only to treatment that leads to a complete cure of the disease, but also to treatment that slows the progression of the disease and / or prolongs the survival of the subject.

[0281] As used in this article, “prevention” means for preventative use (i.e., for subjects susceptible to a given disease) and covers treatment of AML patients with recurrent blastic plasmacytoid dendritic cell tumors (BPDCN).

[0282] As used herein, the term "therapeuticly effective amount" for a multifunctional binding protein or its pharmaceutical composition refers to an antibody-like multifunctional binding protein in an amount sufficient to treat the cancerous disease in a way that provides a reasonable benefit / risk ratio suitable for any pharmaceutical treatment. However, it will be understood that the total daily dosage of the peptides and compositions disclosed herein will be determined by the attending physician within reasonable medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the condition being treated and its severity; the activity of the specific peptide used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific peptide used; the duration of treatment; the drugs used in combination with or concurrently with the specific peptide used; and similar factors well known in the medical field. For example, it is well known within the scope of the art to start the compound dose at a level below that required to achieve the desired therapeutic effect and to gradually increase the dose until the desired effect is achieved.

[0283] As used herein, the terms “subject” or “individual” or “patient” are used interchangeably and may encompass human or non-human mammals, rodents or non-rodents. The terms include, but are not limited to, mammals such as humans (including males, females and children), other primates (monkeys), pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cattle, horses, cats, dogs, sheep, and goats.

[0284] As used herein, the term "pediatric" refers to a pediatric patient aged one month to 18 years. The indicated age should be understood as the age of the pediatric patient at the time of diagnosis of the disease or disorder. Children can be further subdivided into infants (1 to 12 months of age), toddlers aged 1 to 9 years, and older children and adolescents (10 to 18 years). In some embodiments, a pediatric patient may be diagnosed with blastic plasmacytoid dendritic cell neoplasm (BPDCN). In some embodiments, a pediatric patient may be diagnosed with pediatric acute myeloid leukemia (AML). In some embodiments, a pediatric patient may be diagnosed with B-cell acute lymphoblastic leukemia (B-ALL). In some embodiments, a pediatric patient may be diagnosed with high-risk myelodyplasia (HR-MDS). In some embodiments, the pediatric subject is between approximately 1 year and approximately 17 years of age. In some embodiments, the pediatric subject is between approximately 1 year and approximately 11 years of age. In some embodiments, the pediatric subject is approximately 1 year of age. In some embodiments, the pediatric subject is approximately 2 years of age. In some embodiments, the pediatric subjects are approximately 3 years old. In some embodiments, the pediatric subjects are approximately 4 years old. In some embodiments, the pediatric subjects are approximately 5 years old. In some embodiments, the pediatric subjects are approximately 4 years old. In some embodiments, the pediatric subjects are approximately 6 years old. In some embodiments, the pediatric subjects are approximately 7 years old. In some embodiments, the pediatric subjects are approximately 8 years old. In some embodiments, the pediatric subjects are approximately 9 years old. In some embodiments, the pediatric subjects are approximately 10 years old. In some embodiments, the pediatric subjects are approximately 11 years old. In some embodiments, the pediatric subjects are approximately 12 years old. In some embodiments, the pediatric subjects are approximately 13 years old. In some embodiments, the pediatric subjects are approximately 14 years old. In some embodiments, the pediatric subjects are approximately 15 years old. In some embodiments, the pediatric subjects are approximately 16 years old. In some embodiments, the pediatric subjects are approximately 17 years old.

[0285] Unless the context clearly specifies otherwise, as used herein, the singular forms “a,” “an,” and “the” include plural indicators. For example, the term “pharmaceutically acceptable carrier” covers a variety of pharmaceutically acceptable carriers, including mixtures thereof.

[0286] As used in this article, “multiple / various” can therefore include “two / two kinds” or “two or more / two or more kinds”.

[0287] As used herein, "antibody" or "immunoglobulin" can refer to natural or conventional antibodies, in which two heavy chains are linked together by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. Light chains consist of two domains or regions: a variable domain (VL) and a constant domain (CL). Heavy chains typically consist of four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). Specifically, the IgG, IgA, and IgD classes have three heavy chain constant domains, named CH1, CH2, and CH3; and the IgM and IgE classes have four heavy chain constant domains: CH1, CH2, CH3, and CH4. The light chain variable region (VL) and heavy chain variable region (VH) determine antigen binding recognition and specificity. The light chain constant region domain (CL) and heavy chain constant region domain (CH) confer important biological properties, such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to the Fc receptor (FcR). The Fv fragment is the N-terminal portion of the antigen-binding fragment (Fab) of an immunoglobulin and consists of a variable portion of one light chain and one heavy chain.

[0288] Unless otherwise defined, as used herein, when “IgG” or “immunoglobulin G” is generally referred to, it includes IgG1, IgG2, IgG3, and IgG4. In particular, IgG refers to IgG1.

[0289] As used herein, the terms “antibody-like” or “immunoglobulin-like” polypeptides may also refer to unconventional or synthetic antigen-binding polypeptides or binding proteins, including single-domain antibodies and fragments thereof, particularly variable heavy chains of single-domain antibodies, as well as chimeric antibodies, humanized antibodies, bispecific antibodies, or aggregate antibodies.

[0290] As used herein, the term "multifunctional binding protein" encompasses multi-chain proteins, including but not limited to antibody-like peptides or protein forms, comprising at least one first variable region (e.g., a first immunoglobulin heavy chain variable domain (VH) and / or an immunoglobulin light chain variable domain (VL)) specifically binding to a human CD123 peptide and at least one second variable region (e.g., a second immunoglobulin heavy chain variable domain (VH) and / or an immunoglobulin light chain variable domain (VL)) specifically binding to a human NKp46 peptide. While not specifically limited to particular types of constructs, a general embodiment is particularly considered throughout the specification: peptide constructs reported in WO 2015197593 and WO 2017114694 (each of which is incorporated herein by reference). In particular, multifunctional binding proteins (such as those reported in WO 2015197593 and WO 2017114694) can encompass any construct comprising one or more peptide chains.

[0291] As used herein, such as in “humanized antibody,” the term “humanized” refers to a polypeptide (i.e., an antibody or antibody-like polypeptide) that is wholly or partially non-human in origin and has been modified to replace certain amino acids (particularly in the framework regions of the heavy and light chains) in order to avoid or minimize a human immune response. The constant structural domains of humanized antibodies are, in most cases, human CH and CL domains. Many methods for humanizing antibody sequences are known in the art; see, for example, the review by Almagro and Fransson (2008) Front Biosci. 13: 1619-1633. One commonly used method is CDR transplantation or antibody remodeling, which involves transplanting the CDR sequence of a donor antibody (typically a mouse antibody) into the framework scaffold of a human antibody with different specificities.

[0292] For chimeric antibodies, humanization typically involves modifications to the frame region of the variable region sequence. Amino acid residues that are part of the CDR are typically not altered by humanization, although in some cases it may be necessary to modify individual CDR amino acid residues, such as removing glycosylation sites, deamidation sites, or unwanted cysteine ​​residues. N-linked glycosylation occurs by attaching the oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr (where X can be any amino acid other than Pro). Removal of N-glycosylation sites can be achieved by mutating the Asn or Ser / Thr residues to different residues (particularly through conserved substitution). Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly prone to deamidation, primarily when present in the Asn-Gly sequence, and to a lesser extent when present in other dipeptide sequences such as Asn-Ala. When such deamidation sites (especially Asn-Gly) are present in the CDR sequence, it may be necessary to remove these sites, typically through conserved substitution to remove one of the residues involved. Substitution in the CDR sequence to remove one of the residues involved is also intended to be covered by the multifunctional binding protein for which protection is sought.

[0293] As used herein, the term "conservative amino acid substitution" refers to the substitution of an amino acid residue by an amino acid residue with a side chain having similar physicochemical properties. Families of amino acid residues with similar side chains are known in the art and include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Both are acceptable when the amino acid belongs to two different categories (i.e., tyrosine and phenylalanine). Unless otherwise stated, this classification will be followed throughout the specification for reference.

[0294]

[0295] As used herein, the term "domain" can refer to any region of a protein, typically defined based on sequence homology or identity with a particular structural or functional entity. Therefore, the term "region," as used in the context of this disclosure, is broader because it can include additional regions beyond the corresponding domain.

[0296] As used herein, the terms “linker region,” “linker peptide,” “linker polypeptide,” “amino acid linker,” or “linker” refer to any amino acid sequence suitable for covalently linking two polypeptide domains (such as two antigen-binding domains) together and / or covalently linking an Fc region to one or more variable regions (such as one or more antigen-binding domains). While the terms are not limited to a specific size or polypeptide length, such amino acid linkers are typically less than 50 amino acids in length, preferably less than 30 amino acids, for example, 20 amino acids or less, or for example, 15 amino acids or less. Such amino acid linkers may, as desired, comprise all or part of an immunoglobulin polypeptide chain, such as all or part of the hinge region of an immunoglobulin. Alternatively, amino acid linkers may comprise polypeptide sequences not derived from the hinge region of an immunoglobulin (or even not derived from the heavy or light polypeptide chain of an immunoglobulin).

[0297] As used in this article, immunoglobulin hinge regions or fragments thereof can therefore be considered as a specific type of linker derived from immunoglobulin polypeptide chains.

[0298] As used herein, the term "hinge region" or "hinge" refers to a generally flexible region derived from the corresponding heavy chain polypeptide, which separates the Fc and Fab portions of certain isotypes of immunoglobulins (more particularly IgG, IgA, or IgD isotypes). Such hinge regions are known in the art and depend on the isotype of the immunoglobulin under consideration. For native IgG, IgA, and IgD isotypes, the hinge region thus separates the CH1 and CH2 domains and is typically cleaved after papain digestion. On the other hand, the regions corresponding to the hinge in the IgM and IgE heavy chains are typically formed by additional, less flexible, constant domains. Furthermore, the hinge region may contain one or more cysteine ​​residues involved in interchain disulfide bonds. When applicable, the hinge region may also contain one or more Fcγ receptor binding sites in addition to the FcγR binding site derived from the CH2 domain. Furthermore, the hinge region may contain one or more post-translational modifications, such as one or more glycosylated residues, depending on the isotype under consideration. Therefore, it will be readily understood that the term "hinge" as used throughout the specification is not limited to a specific group of hinge sequences or a specific location on a structure. Unless otherwise stated, the hinge region still particularly considered includes all or part of the hinge from an immunoglobulin belonging to one of the following isotypes: IgG isotype, IgA isotype, and IgD isotype; especially the IgG isotype.

[0299] As used herein, the terms “CH domain” or “CH domain” or “constant domain” are used interchangeably and refer to any one or more heavy chain immunoglobulin constant domains. Such CH domains naturally fold into immunoglobulin-like domains, although they may be partially disordered in a dissociated form (e.g., the CH1 domain when not associated with a light chain constant domain (CL)). Unless otherwise stated, the terms may therefore refer to the CH1 domain, the CH2 domain, the CH3 domain; or any combination thereof.

[0300] As used herein, the terms “CH1 domain” or “CH1 domain” or “constant domain 1” are used interchangeably and refer to the corresponding heavy chain immunoglobulin constant domain 1.

[0301] As used herein, the terms “CH2 domain” or “CH2 domain” or “constant domain 2” are used interchangeably and refer to the corresponding heavy chain immunoglobulin constant domain 2.

[0302] As used herein, the terms “CH3 domain” or “CH3 domain” or “constant domain 3” are used interchangeably and refer to the corresponding heavy chain immunoglobulin constant domain 3.

[0303] As used herein, such as in (CH2-CH3)A and (CH2-CH3)B, the term "CH2-CH3" therefore refers to a polypeptide sequence containing immunoglobulin heavy chain constant domain 2 (CH2) and immunoglobulin heavy chain constant domain 3 (CH3).

[0304] As used herein, the terms “CL domain” or “CL domain” are used interchangeably and refer to the corresponding light chain immunoglobulin constant domain. Unless otherwise stated, this term can therefore cover the CL domain of immunoglobulin light chains of the kappa (κ or K) or lambda (λ) classes (including all known subtypes (e.g., λ1, λ2, λ3, and λ7)). In particular, when the CL domain belongs to the κ class, it may also be referred to herein as the Cκ or CK or Ck domain.

[0305] As used herein, the term "paired C(CH1 / CL)" or "paired C(CH1 / CL)" refers to a constant heavy chain domain 1 and a constant light chain domain (e.g., a light chain of an immunoglobulin of the kappa (κ or K) or lambda (λ) class) bonded together by covalent or non-covalent bonds (preferably non-covalent bonds); thereby forming a heterodimer. Unless otherwise stated, this term can therefore cover all possible combinations when the pairing constant chain domains are not present on the same polypeptide chain. Preferably, the corresponding CH1 and CL domains will therefore be selected to be complementary to each other, such that they form a stable paired C(CH1 / CL).

[0306] Advantageously, when the binding protein comprises multiple paired C domains, such as a "pair of C1 (CH1 / CL)" and a "pair of C2 (CH1 / CL)," each CH1 and CL domain forming the pair is selected such that they are formed between complementary CH1 and CL domains. Embodiments of complementary CH1 and CL domains have previously been described in international patent applications WO 2006064136, WO2012089814, or WO 2015197593A1.

[0307] Unless otherwise stated, the terms "pair C1 (CH1 / CL)" or "pair C2 (CH1 / CL)" may refer to different constant pair domains (C1 and C2) formed by the same or different constant heavy 1-domains (CH1) and the same or different constant light chain domains (CL). Preferably, the terms "pair C1 (CH1 / CL)" or "pair C2 (CH1 / CL)" may refer to different constant pair domains (C1 and C2) formed by the same constant heavy 1-domains (CH1) and the same constant light chain domains (CL).

[0308] As used herein, the terms "Fc region" or "crystallizable fragment region" or alternatively "Fc portion" encompass all or part of the "Fc domain," which may therefore include an immunoglobulin hinge region (naturally bearing a first binding site for FcγR), a CH2 domain (naturally bearing a second binding site for FcγR), and the CH3 domain of an immunoglobulin (e.g., IgG, IgA, or IgD immunoglobulin) and / or (where applicable) the CH4 domain of an immunoglobulin (e.g., for IgM and IgE). Preferably, the Fc region includes at least all or part of the CH2 and CH3 domains, and, if necessary, all or part of the immunoglobulin hinge region. The term may therefore refer to molecules (whether in monomeric or aggregate form) containing sequences of non-antigen-binding fragments produced by antibody digestion or by other means, and may contain a hinge region. The original immunoglobulin source of the native Fc is particularly human-derived and may be any immunoglobulin, although IgG1 is preferred. Natural Fc molecules consist of monomeric polypeptides that can be covalently (i.e., disulfide bonds) and non-covalently linked into dimers or aggregates. Depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgGA1, and IgGA2), the number of intermolecular disulfide bonds between the monomeric subunits of a natural Fc molecule ranges from 1 to 13. One embodiment of natural Fc is a disulfide-bonded dimer produced by papain digestion of IgG. The term "natural Fc" as used herein is general for monomeric, dimeric, and aggregate forms. Under this term, the "Fc region" may therefore comprise, or consist of, all or part of, CH2-CH3 (e.g., (CH2-CH3)A or (CH2-CH3)B or their binding pairs) and, if desired, a binding site for human FcγR on an immunoglobulin hinge region. Unless otherwise stated, the term "Fc region" may refer to a natural or variant Fc region.

[0309] As used herein, the term "Fc variant" refers to a molecule or sequence modified from a natural Fc but still containing a binding site for the receptor FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with receptors are known in the art. Thus, the term "Fc variant" can comprise molecules or sequences humanized from non-human natural Fc. Furthermore, natural Fc contains regions that can be removed because they provide structural features or biological activities not required by the antibody-like binding proteins of the present invention. Thus, the term "Fc variant" includes molecules or sequences lacking one or more natural Fc sites or residues or where one or more Fc sites or residues have been modified, said one or more Fc sites or residues affecting or involved in: (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) N-terminal heterogeneity when expressed in selected host cells, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than rescue receptors, or (7) antibody-dependent cytotoxicity (ADCC).

[0310] According to this disclosure, the crystallizable fragment (Fc) region (e.g., native or variant) retains the ability to bind to the human Fc-γ receptor polypeptide (Fcγ), which typically appears on the native Fc region via antibody Fc-hinge region binding. For reference, IgG1, IgG2, and IgG4 have similar overall structures and more than 90% sequence homology, with the main differences existing in the hinge region and CH2 domain (which forms the main binding site for FcγR). The hinge region also functions as a flexible linker between the Fab and Fc moieties.

[0311] An Fc region having one or more amino acid modifications (e.g., substitution, deletion, insertion) in one or more portions is still considered an Fc region, which increases the affinity and coercivity of the variant Fc region to FcγRs (including activating and inhibitory FcγRs). In some embodiments, the one or more amino acid modifications increase the affinity of the Fc region to FcγRIIIA and / or FcγRIIA. In another embodiment, the variant Fc region further binds specifically to FcγRIIB with a lower affinity than the Fc region of a reference parent antibody (e.g., an antibody having the same amino acid sequence as the antibody except for one or more amino acid modifications in the Fc region). Therefore, the natural and variant Fc regions considered herein generally contain a domain capable of binding to human CD16 (i.e., a CH2 domain), such as an N-linked glycosylated human Fc domain containing amino acid residue N297 (according to EU numbering).

[0312] As used herein, the term "Fc-competent" therefore refers to a binding protein that can specifically bind to FcγR (especially activated FcγR), particularly to one of FcγRI (CD64a), FcγRIIa (CD32a) and FcγRIIIa (CD16a), and more particularly to FcγRIIIa (CD16a).

[0313] Alternatively, several modifications have been reported to directly affect binding to FcγR, including mutations at residue 297 (according to EU numbering) or, alternatively, mutations at residues 234 and 235 (according to the EU numbering system) in the lower hinge region.

[0314] As used herein, the term “Fc silencing” refers to a binding protein having an Fc region that lacks a binding site for FcγR (e.g., an Fc region lacking a CH2 domain with the binding site and a hinge region with the binding site); particularly FcγRI, FcγRIIa, and FcγRIIIa, and more particularly the binding site for FcγRIIIa (CD16a).

[0315] As used herein, such as in the term "variable domain," the term "variable" refers to certain portions of the associated binding protein that differ widely in sequence between two antibodies and among multiple antibodies, and are used for the specific recognition and binding of a particular antibody to its specific target. However, variability is not uniformly distributed throughout the entire variable domain of an antibody. Variability is concentrated in three segments called complementarity-determining regions (CDRs; namely CDR1, CDR2, and CDR3) (also known as highly variable regions), in both the light chain and heavy chain variable domains. The more highly conserved portions of the variable domain are called frame (FR) regions or sequences.

[0316] As used herein, the terms “VH domain” or “VH domain” are used interchangeably and refer to the corresponding heavy chain immunoglobulin variable domain.

[0317] As used herein, the terms “VL domain” or “VL domain” are used interchangeably and refer to the corresponding light chain immunoglobulin variable domain.

[0318] When the VH or VL domains associate with the first antigen-binding domain (ABD) or the second antigen-binding domain, they may also be referred to as “VH1” and “VL1” or “VH2” and “VL2”, respectively, in this document.

[0319] The terms “binding pair V (VH / VL)”, “VH / VL pair”, “(VH / VL) pair”, “VL / VH pair”, or “(VL / VH) pair” are used interchangeably. Heavy chain variable domains and light chain variable domains may pair in parallel to form antigen-binding domains (ABDs). Each binding pair includes both a VH region and a VL region. Unless otherwise stated, the terms do not specify which immunoglobulin variable regions are VH or VL regions or which ABDs will specifically bind to proteins expressed on the surface of immune effector cells or target cells (e.g., NKp46 and CD123).

[0320] As used herein, the term "highly variable region" refers to the amino acid residues in the antibody responsible for antigen binding. This term may be replaced by the terms "complementarity-determining region" or "CDR".

[0321] Therefore, as used herein, the “complementarity-determining region” or “CDR” refers to the amino acid sequence that commonly determines the binding affinity and specificity of the native Fv region, which is the natural immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, named CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3, respectively. Thus, the conventional antibody-antigen binding domain comprises six CDRs, which contain sets of CDRs from the various variable regions of the heavy and light chains. Furthermore, as used herein, the “frame region” (FR) refers to the amino acid sequence between CDRs, i.e., those portions of the relatively conserved variable regions of the immunoglobulin light and heavy chains across different immunoglobulins within a single species. The light and heavy chains of immunoglobulins each have four FRs, named FR-L1, FR-L2, FR-L3, FR-L4 and FR-H1, FR-H2, FR-H3, FR-H4, respectively. Therefore, the variable structural domain of the light chain can be named (FR-L1)-(CDR-L1)-(FR-L2)-(CDR-L2)-(FR-L3)-(CDR-L3)-(FR-L4), and the variable structural domain of the heavy chain can be named (FR-H1)-(CDR-H1)-(FR-H2)-(CDR-H2)-(FR-H3)-(CDR-H)-(FR4-H3).

[0322] The highly variable region typically contains amino acid residues from the “complementarity-determining region” or “CDR” (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al. 1991) and / or residues from the “highly variable loop” (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol [Journal of Molecular Biology] 1987;196:901-917). The amino acid residues in this region are numbered according to the method described above by Kabat et al. Therefore, phrases such as “Kabat position,” “variable domain residue number as in Kabat,” and “according to Kabat” in this document refer to this numbering system used for heavy chain or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids, corresponding to a shortening or insertion of the FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion after residue 52 of CDR H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat number of residues for a given antibody can be determined by comparing homologous regions of the antibody sequence with sequences numbered according to “standard” Kabat.

[0323] Where necessary, the CDR is defined as such by EU, Kabat, Chotia, or IMGT numbering. The correspondences between these classifications are known in the art, as further detailed by reference to IMGT® or the International ImMunoGeneTics Information System® (CNRS and Montpellier University) and as Lefranc (Biomolecules; 2014; 4, 1102-1139) and Dondelinger (Frontiers in Immunology; 2018; 9, 2278). The CDR may also be defined according to the Honegger-Pluckthun (“Honegger”) numbering scheme as described in Honegger and Pluckthun (2001), J. Mol. Biol., Vol. 309(3): 657-670.

[0324] Unless otherwise stated, residue numbering in this paper will refer to EU, Kabat, Chotia, IMGT, or Honegger-Pluckthun numbering conventions. In case of conflict regarding the exact location of highly variable regions within the reference sequence, the Kabat numbering convention will prevail. In case of conflict regarding the exact location of constant regions within the reference sequence, the EU numbering convention will prevail.

[0325] As used herein, the term "cytotoxicity" refers to the toxicity of a compound (such as the multifunctional binding protein according to this disclosure) to tumor cells. Cytotoxicity can be induced by different mechanisms of action and can therefore be classified as cell-mediated cytotoxicity, apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

[0326] As used herein, the terms “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refer to cell-mediated immune defense mechanisms in which effector cells of the immune system actively lyse target cells whose membrane surface antigens have been bound by the specific antibodies or multifunctional binding proteins disclosed herein.

[0327] As used herein, the terms “proliferative disorder,” “hyperproliferative disorder,” and / or “cancer” refer not only to solid tumors such as breast cancer, respiratory cancer, brain cancer, reproductive organ cancer, digestive tract cancer, urinary tract cancer, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer, and their distant metastases, but also to blood cancers, including tumors of hematopoietic and lymphoid tissues such as lymphoma, myeloma, and leukemia. Leukemia includes, but is not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.

[0328] As used in this article, "acute myeloid leukemia (AML)" is a clonal disorder clinically characterized by increased proliferation of heterogeneous and undifferentiated myeloid-blast cells. Not to be bound by theory, leukemia grade is maintained by a small population of LSCs (leukemia stem cells) (AML-LSCs) (which possess unique self-renewal capacity and can differentiate into leukemia precursor cells). These precursor cells produce a large number of leukemia blast cells, readily detectable in patients at diagnosis and relapse, ultimately leading to death. AML-LSCs are typically reported as quiescent cells compared to rapidly dividing clonal precursor cells.

[0329] In the context of AML, the term "relapse" can be specifically defined as a relapse of AML after complete remission. In this sense, "complete remission" or "CR" can be defined as follows: normal neutrophil count (> 1.0*10⁹ / L), normal heme level (10 g / dL), and normal platelet count (> 100*10⁹ / L), without dependence on red blood cell transfusion; less than 5% blastocytes, no blastocyte clusters or aggregates, and no Ossne bodies on bone marrow examination; and normal blood cell maturation (morphology; myelography) and the absence of extramedullary leukemia.

[0330] As used in this article, the term "refractory" refers to cancer that does not respond to treatment. In the context of AML, most patients achieve remission (absence of signs and symptoms) after initial treatment. However, some patients still have residual leukemia cells in their bone marrow even after intensive therapy. Patients who have not achieved complete remission after two cycles of induction chemotherapy are typically diagnosed with "refractory AML".

[0331] As used herein, the term "blastic plasmacytoid dendritic cell tumor" or "BPDCN" refers to a rare hematologic malignancy characterized by plasmacytoid dendritic cells. In 2016, the World Health Organization (WHO) designated BPDCN as its own category within the myeloid category of tumors. It is estimated that BPDCN accounts for approximately 0.44% of all hematologic malignancies. BPDCN is an aggressive malignancy characterized by cutaneous lymphoma and / or leukemia. Particularly in later stages, the disease may also involve malignant plasmacytoid dendritic cell infiltration in the liver, spleen, lymph nodes, central nervous system, or other tissues, thereby damaging these organs. The tumor forms in individuals of all ages. In children, it affects both men and women equally, but in adults, it is more common in men (approximately 75% of cases). Almost all cases of BPDCN involve cells that overexpress CD123.

[0332] As used herein, “myelodysplastic syndromes” (“MDS”) (formerly known as preleukemia) are a collection of blood disorders involving the ineffective production (or dysplasia) of bone marrow-based blood cells. They represent a range of clonal hematopoietic stem cell disorders characterized by progressive bone marrow failure and an increased risk of progression to acute myeloid leukemia (“AML,” also known as acute myeloid leukemia). The International Prognostic Scoring System (IPSS) is widely used to identify patients with high-risk characteristics based on the severity of blood cell reduction, the percentage of myeloblasts in the bone marrow, and cytogenetic abnormalities.

[0333] As used herein, “pharmaceutically acceptable carrier” is intended to include any and all carriers (such as any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and absorption delay agents) compatible with drug administration, particularly parenteral administration. The use of such media and reagents for the active pharmaceutical ingredient is known. Such media may be used in the compositions of this disclosure unless any conventional media or reagent is incompatible with the active compound. For example, formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Embodiments of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffer media. In a non-exhaustive manner, pharmaceutically acceptable carriers include, but are not limited to, 0.01–0.1 M (e.g., 0.05 M) phosphate buffer or 0.8% saline. Other common parenteral media include sodium phosphate solution, Ringer's dextran, dextran and sodium chloride, lactated Ringer's solution, or fixed oil. Intravenous media include fluids and nutritional supplements, electrolyte supplements, such as those based on Ringer's dextran. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases. More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (in the case of water solubility) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be a fluid present to a degree of ease of injection. It should be stable under manufacturing and storage conditions and preserved against contamination by microorganisms (such as bacteria and fungi) in the embodiments. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings (such as lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants. Antimicrobial activity can be achieved using various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In some embodiments, the composition includes isotonic agents such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride. Extended absorption of the injectable composition can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0334] As used herein and unless otherwise stated, the term "at least one / at least one" may cover "one or more / one or more types" or even "two or more / two or more types" (or "multiple / various types"). For example, it may cover 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 5 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or greater than 100.

[0335] As used herein and unless otherwise stated, the term "less than..." can cover all values ​​from 0 to the corresponding threshold. For example, when applicable, it can cover values ​​less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or less than 100.

[0336] As used herein, the term "cell" can encompass any prokaryotic or eukaryotic cell. Cell types particularly considered are those suitable for the production and / or engineering of recombinant antibodies or their fragments or polypeptide chains. Such cells can be selected from the following groups in a non-exhaustive manner: bacterial cells, yeast cells, mammalian cells, non-mammalian cells, insect cells, and plant cells.

[0337] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny cells of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their progeny, regardless of passage number. Progeny cells may not be identical to parent cells in terms of nucleic acid content, but may contain mutations. This document includes mutant progeny having the same function or biological activity as screened or selected in the initially transformed cells. Host cell lines can be any type of cell system used to produce the binding proteins of this disclosure. Host cells can therefore include cultured cells, such as mammalian cultured cells (e.g., CHO cells, HEK cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells), bacterial cells, yeast cells, insect cells, and plant cells, to name just a few.

[0338] "Isolated" nucleic acid molecules or polynucleotides refer to nucleic acid molecules, DNA or RNA, that have been removed from their natural environment. For example, for the purposes of this disclosure, recombinant polynucleotides encoding polypeptides contained in a vector are considered isolated. Other embodiments of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or purified (partial or substantially) polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that typically contain polynucleotide molecules, but said polynucleotide molecules are present outside the chromosome or at chromosomal locations different from their natural chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this disclosure, as well as positive-strand and negative-strand forms and double-strand forms. Isolated polynucleotides or nucleic acids according to this disclosure further include synthetically produced such molecules. In addition, polynucleotides or nucleic acids may be or may include regulatory components such as promoters, ribosome binding sites, or transcription terminators.

[0339] The term “vector” or “expression vector” is intended to refer to a medium through which nucleic acid, particularly DNA or RNA sequences (e.g., foreign genes) can be introduced into a host cell in order to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence.

[0340] II. Binding proteins

[0341] This document discloses a binding protein for use in methods of treating leukemia and myelodysplastic syndromes, the binding protein comprising a bispecific NK cell binder (NKCE) having an active Fc domain capable of binding to CD16a (FcγRIIIa). The disclosed NKCE functions as a trifunctional molecule binding to NKp46 and CD16a on the surface of NK cells and CD123 on malignant cells. The co-binding of the disclosed CD123 NKCE to NK cells and malignant cells leads to the formation of immune synapses that induce NK cell activation and degranulation.

[0342] In some embodiments, the binding protein is characterized by comprising a first antigen-binding domain specific for binding to CD123 and a second antigen-binding domain specific for binding to NKp46. In some embodiments, the binding protein comprises a third antigen-binding domain for binding to CD16. In some embodiments, the third antigen-binding domain for binding to CD16 is an antigen-binding domain specific for binding to CD16. In some embodiments, the third antigen-binding domain is an Fc domain.

[0343] In some embodiments, the NKCE is characterized in that it comprises a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46, wherein the first antigen-binding domain comprises:

[0344] The heavy chain variable domain (VH1) comprises CDR-H1, H2, and H3 corresponding to amino acid sequences of SEQ ID NO: 1, 2, and 3, or corresponding to amino acid sequences of SEQ ID NO: 4, 5, and 5, respectively; and

[0345] The light chain variable domain (VL1) contains CDR-L1, L2 and L3 corresponding to amino acid sequences of SEQ ID NO: 7, 8 and 9, or amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively.

[0346] In some embodiments, the binding protein is characterized in that the first antigen-binding domain having binding specificity comprises:

[0347] The heavy chain variable domain (VH1) comprises CDR-H1, H2, and H3 corresponding to amino acid sequences of SEQ ID NO: 1, 2, and 3, or corresponding to amino acid sequences of SEQ ID NO: 4, 5, and 5, respectively; and

[0348] The light chain variable domain (VL1) contains CDR-L1, L2 and L3 corresponding to amino acid sequences of SEQ ID NO: 7, 8 and 9, or amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively.

[0349] In some embodiments, the binding protein is characterized in that it comprises VH1 containing at least about 90% of the amino acid sequence identical to that of SEQ ID NO:41, and wherein VL1 contains at least about 90% of the amino acid sequence identical to that of SEQ ID NO:43; or VH1 containing at least about 90% of the amino acid sequence identical to that of SEQ ID NO:42, and wherein VL1 containing at least about 90% of the amino acid sequence identical to that of SEQ ID NO:44.

[0350] In some embodiments, the binding protein is characterized in that it comprises the amino acid sequence of SEQ ID NO:41 for VH1 and the amino acid sequence of SEQ ID NO:43 for VL1; or the amino acid sequence of SEQ ID NO:42 for VH1 and the amino acid sequence of SEQ ID NO:44 for VL1.

[0351] In some embodiments, the binding protein includes a second antigen-binding domain that is specific for binding to NKp46. In some embodiments, the second antigen-binding domain includes a CDR defined by the Kabat designation. In some embodiments, the second antigen-binding domain includes a CDR defined by the IMGT designation. In some embodiments, the second antigen-binding domain includes a CDR defined by the Chothia designation. In some embodiments, the second antigen-binding domain includes a CDR defined by the Honegger designation. In some embodiments, the second antigen-binding domain includes a CDR as defined in Table 1.

[0352] Table 1. Examples of anti-NKp46 CDR

[0353]

[0354] In some embodiments, the binding protein is characterized in that the second antigen-binding domain having binding specificity to NKp46 comprises:

[0355] a. The second heavy chain variable domain (VH2) contains CDRs H1, H2, and H3 corresponding to the following amino acid sequences:

[0356] i. Specifically, SEQ ID NO: 13, 14 and 15;

[0357] ii. Specifically, SEQ ID NO: 16, 17, and 18;

[0358] iii. Specifically, SEQ ID NO: 19, 20, and 21;

[0359] iv. respectively, SEQ ID NO: 22, 23 and 24; or

[0360] v. respectively, SEQ ID NO: 16, 25 and 26; and

[0361] b. The second light chain variable domain (VL2) contains CDRs-L1, L2, and L3 corresponding to the following amino acid sequences:

[0362] i. Specifically, SEQ ID NO: 27, 28, and 29;

[0363] ii. Specifically, SEQ ID NO: 30, 31 and 32;

[0364] iii. Specifically, SEQ ID NO: 33, 34 and 35;

[0365] iv. respectively, SEQ ID NO: 36, 37 and 38; or

[0366] v. respectively, SEQ ID NO: 39, 31 and 40.

[0367] In some embodiments, the binding protein is characterized by:

[0368] a. The VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 45, and the VL1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 53; or

[0369] b. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 46, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 54;

[0370] c. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 47, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 55;

[0371] d. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 48, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 56;

[0372] e. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 49, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 57;

[0373] f. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 50, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 58;

[0374] g. The VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 51, and wherein the VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 59; or

[0375] h. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 52, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 60.

[0376] In some embodiments, the binding protein is characterized by:

[0377] a. The VH2 comprises the amino acid sequence of SEQ ID NO: 45, and the VL1 comprises the amino acid sequence of SEQ ID NO: 53; or

[0378] b. The VH2 comprises the amino acid sequence of SEQ ID NO: 46, and the VL2 comprises the amino acid sequence of SEQ ID NO: 54;

[0379] c. The VH2 comprises the amino acid sequence of SEQ ID NO: 47, and the VL2 comprises the amino acid sequence of SEQ ID NO: 55;

[0380] d. The VH2 comprises the amino acid sequence of SEQ ID NO: 48, and the VL2 comprises the amino acid sequence of SEQ ID NO: 56;

[0381] e. The VH2 comprises the amino acid sequence of SEQ ID NO: 49, and the VL2 comprises the amino acid sequence of SEQ ID NO: 57;

[0382] f. The VH2 comprises the amino acid sequence of SEQ ID NO: 50, and the VL2 comprises the amino acid sequence of SEQ ID NO: 58;

[0383] g. The VH2 comprises the amino acid sequence of SEQ ID NO: 51, and the VL2 comprises the amino acid sequence of SEQ ID NO: 59; or

[0384] h. The VH2 contains the amino acid sequence of SEQ ID NO: 52, and the VL2 contains the amino acid sequence of SEQ ID NO: 60.

[0385] In some embodiments, the binding protein is characterized in that it comprises three polypeptide chains (I), (II), and (III) forming two ABDs, as defined below:

[0386] V 1A - C 1A - Hinge 1 - (C H 2-C H 3) A (I)

[0387] V 1B - C 1B - Hinge 2 - (C H 2-C H 3) B - L1 - V 2A - C 2A - Hinge 3(II)

[0388] V 2B - C 2B (III)

[0389] in:

[0390] V 1A and V 1B Forming binding pairs V1 (V H1 / V L1 );

[0391] V 2A and V 2B Forming binding pairs V2 (V H2 / V L2 );

[0392] C1A and C 1B Forming a pair of C1 (C H 1 / C L ), and C 2A and C 2B Formation of C2 (C H 1 / C L ), where C H 1 represents the immunoglobulin heavy chain constant domain 1, and C L It is the constant structural domain of the immunoglobulin light chain;

[0393] Hinge 1, hinge 2 and hinge 3 may be the same or different, and correspond to all or part of the immunoglobulin hinge region;

[0394] (C H 2-C H 3) A and (C) H 2-C H 3) B For the same or different, and containing the immunoglobulin heavy chain constant domain 2 (C H 2) and immunoglobulin heavy chain constant domain 3 (C H 3);

[0395] L1 is an amino acid linker.

[0396] In some embodiments, the binding protein is characterized by containing C 1B For immunoglobulin heavy chain constant domain 1 (C H 1);

[0397] C 2A For immunoglobulin heavy chain constant domain 1 (C H 1);

[0398] C L Corresponding to the immunoglobulin κ light chain constant domain (C κ );

[0399] (C H 2-C H 3) A The amino acid sequence corresponding to SEQ ID NO: 69;

[0400] (C H 2-C H 3) B The amino acid sequence corresponding to SEQ ID NO: 70;

[0401] Hinge 1 corresponds to the amino acid sequence of SEQ ID NO: 74;

[0402] Hinge 2 corresponds to the amino acid sequence of SEQ ID NO: 75;

[0403] Hinge 3 corresponds to the amino acid sequence of SEQ ID NO: 77;

[0404] L1 corresponds to the amino acid sequence of SEQ ID NO: 76.

[0405] In some embodiments, the binding protein is characterized in that residue N297 of the Fc region or a variant thereof, according to the EU number, contains an N-linked glycosylation.

[0406] In some embodiments, the binding protein is characterized in that all or part of the Fc region or a variant thereof binds to the human CD16A (FcγRIII) polypeptide.

[0407] In some embodiments, the binding protein is characterized in that at least two polypeptide chains are linked by at least one disulfide bond.

[0408] In some embodiments, the binding protein is characterized in that the polypeptide chain (I) and the polypeptide chain (II) are connected via C 1A At least one disulfide bond is connected between hinge 2 and the polypeptide chain (II) and the polypeptide chain (III) via hinge 3 to C. 2B At least one disulfide bond connects them.

[0409] In some embodiments, the binding protein is characterized by V 1A For V L1 And V 1B For V H1 .

[0410] In some embodiments, the binding protein is characterized by V 2A For V H2 And V 2B For V L2 .

[0411] In some embodiments, the binding protein is characterized by:

[0412] (a) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 13; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 14; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 15; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 27; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 28; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 29.

[0413] (b) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 17; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 18; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 30; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 32.

[0414] (c) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2The CDR-H1 contains the amino acid sequence of SEQ ID NO: 19; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 20; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 21; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 33; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 34; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 35.

[0415] (d) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 22; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 23; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 24; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 36; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 37; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 38.

[0416] (e) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 25; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 26; V L2The CDR-L1 contains the amino acid sequence of SEQ ID NO: 39; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 40.

[0417] (f) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 13; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 14; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 15; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 27; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 28; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 29.

[0418] (g) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 17; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 18; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 30; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 32.

[0419] (h) V H1The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 19; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 20; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 21; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 33; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 34; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 35.

[0420] (i) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 22; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 23; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 24; V L2 The CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; the CDR-L2 containing the amino acid sequence of SEQ ID NO: 37; the CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; or

[0421] (j) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1CDR-L1 containing the amino acid sequence of SEQ ID NO: 10; CDR-L2 containing the amino acid sequence of SEQ ID NO: 11; CDR-L3 containing the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 25; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 26; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 39; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO: 40.

[0422] In some embodiments, the binding protein is characterized by:

[0423] (a) V H1 and V L1 The amino acid sequences corresponding to SEQ ID NO: 41 and 43, or the amino acid sequences corresponding to SEQ ID NO: 42 and 44, respectively;

[0424] and / or

[0425] (b) V H2 and V L2 Corresponding to

[0426] The amino acid sequences of SEQ ID NO: 45 and 53;

[0427] The amino acid sequences of SEQ ID NO: 46 and 54;

[0428] The amino acid sequences of SEQ ID NO: 47 and 55;

[0429] The amino acid sequences of SEQ ID NO: 48 and 56;

[0430] The amino acid sequences of SEQ ID NO: 49 and 57;

[0431] The amino acid sequences of SEQ ID NO: 50 and 58;

[0432] The amino acid sequences of SEQ ID NO: 51 and 59; or

[0433] The amino acid sequences of SEQ ID NO: 52 and 60.

[0434] In some embodiments, the binding protein is characterized by:

[0435] (a)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 45; V L2 Contains the amino acid sequence of SEQ ID NO: 53;

[0436] (b)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 46; V L2 The amino acid sequence containing SEQ ID NO: 54;

[0437] (c)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 47; V L2 Contains the amino acid sequence of SEQ ID NO: 55;

[0438] (d)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 48; V L2 Contains the amino acid sequence of SEQ ID NO: 56;

[0439] (e)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 49; VL2 contains the amino acid sequence of SEQ ID NO: 57.

[0440] (f)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 50; V L2The amino acid sequence containing SEQ ID NO: 58;

[0441] (g)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 51; V L2 Contains the amino acid sequence of SEQ ID NO: 59;

[0442] (h)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 52; VL2 contains the amino acid sequence of SEQ ID NO: 60.

[0443] (i)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 45; VL2 contains the amino acid sequence of SEQ ID NO: 53.

[0444] (j)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 46; V L2 The amino acid sequence containing SEQ ID NO: 54;

[0445] (k)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 47; V L2 Contains the amino acid sequence of SEQ ID NO: 55;

[0446] (l)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2Contains the amino acid sequence of SEQ ID NO: 48; V L2 Contains the amino acid sequence of SEQ ID NO: 56;

[0447] (m)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 49; V L2 The amino acid sequence containing SEQ ID NO: 57;

[0448] (n)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 50; V L2 The amino acid sequence containing SEQ ID NO: 58.

[0449] (o)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 51; V L2 Contains the amino acid sequence of SEQ ID NO: 59;

[0450] (p)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 52; V L2 It contains the amino acid sequence of SEQ ID NO: 60.

[0451] In some embodiments, the binding protein is characterized by:

[0452] -The polypeptide (I) consists of the amino acid sequence of SEQ ID NO: 64;

[0453] -The polypeptide (II) consists of the amino acid sequence of SEQ ID NO: 65; and

[0454] -The polypeptide (III) consists of the amino acid sequence of SEQ ID NO: 66.

[0455] III. Use / Treatment Methods

[0456] This article provides a method for treating or preventing blood disorders or disorders in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the CD123 NKCE of this disclosure.

[0457] This article provides a method for treating or preventing leukemia or myelodysplastic syndrome or BPDCN in a subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46.

[0458] In some embodiments, the subject in need is an adult. In some embodiments, the adult is a subject over 17 years of age. In some embodiments, the adult is a subject over 18 years of age.

[0459] In some embodiments, the subjects in need are pediatric subjects. In some embodiments, the pediatric subjects are between about 1 year and about 17 years old. In some embodiments, the pediatric subjects are between about 1 year and about 11 years old.

[0460] The therapeutically effective dose of the NK cell binder disclosed herein can be a dose or amount sufficient to induce a “therapeutic response” in a subject, said therapeutic response being an improvement in at least one measure of a blood disorder or disorder (e.g., ALL, B-ALL, BPDCN, or HR-MDS). In some embodiments, the therapeutically effective dose is expressed as µg / kg patient body weight (µg / kg).

[0461] In some embodiments, the effective therapeutic dose is between about 1 µg / kg and about 6000 µg / kg. In some embodiments, the effective therapeutic dose is between about 1 µg / kg and about 10 µg / kg. In some embodiments, the effective therapeutic dose is between about 10 and about 100 µg / kg. In some embodiments, the effective therapeutic dose is between about 100 µg / kg and about 150 µg / kg. In some embodiments, the effective therapeutic dose is between about 150 µg / kg and about 200 µg / kg. In some embodiments, the effective therapeutic dose is between about 200 µg / kg and about 250 µg / kg. In some embodiments, the effective therapeutic dose is between about 250 µg / kg and about 300 µg / kg. In some embodiments, the effective therapeutic dose is between about 300 µg / kg and about 350 µg / kg. In some embodiments, the effective therapeutic dose is between about 350 µg / kg and about 400 µg / kg. In some embodiments, the effective therapeutic dose is between about 400 µg / kg and about 450 µg / kg. In some embodiments, the effective therapeutic dose is between about 450 µg / kg and about 500 µg / kg. In some embodiments, the effective therapeutic dose is between about 500 µg / kg and about 550 µg / kg. In some embodiments, the effective therapeutic dose is between about 550 µg / kg and about 600 µg / kg. In some embodiments, the effective therapeutic dose is between about 600 µg / kg and about 650 µg / kg. In some embodiments, the effective therapeutic dose is between about 650 µg / kg and about 700 µg / kg. In some embodiments, the effective therapeutic dose is between about 700 µg / kg and about 750 µg / kg. In some embodiments, the effective therapeutic dose is between about 750 µg / kg and about 800 µg / kg. In some embodiments, the effective therapeutic dose is between about 800 µg / kg and about 850 µg / kg. In some embodiments, the effective therapeutic dose is between about 850 µg / kg and about 900 µg / kg. In some embodiments, the effective therapeutic dose is between about 900 µg / kg and about 950 µg / kg. In some embodiments, the effective therapeutic dose is between about 950 µg / kg and about 1000 µg / kg. In some embodiments, the effective therapeutic dose is between about 1000 µg / kg and about 1300 µg / kg. In some embodiments, the effective therapeutic dose is between about 1300 µg / kg and about 1500 µg / kg. In some embodiments, the effective therapeutic dose is between about 1500 µg / kg and about 2000 µg / kg.In some embodiments, the effective therapeutic dose is between about 2000 µg / kg and about 2500 µg / kg. In some embodiments, the effective therapeutic dose is between about 2500 µg / kg and about 3000 µg / kg. In some embodiments, the effective therapeutic dose is between about 3000 µg / kg and about 4000 µg / kg. In some embodiments, the effective therapeutic dose is between about 4000 µg / kg and about 4500 µg / kg. In some embodiments, the effective therapeutic dose is between about 4500 µg / kg and about 5000 µg / kg. In some embodiments, the effective therapeutic dose is between about 5000 µg / kg and about 6000 µg / kg.

[0462] In some embodiments, the effective therapeutic dose is about 3 µg / kg. In some embodiments, the effective therapeutic dose is about 10 µg / kg. In some embodiments, the effective therapeutic dose is about 13 µg / kg. In some embodiments, the effective therapeutic dose is about 15 µg / kg. In some embodiments, the effective therapeutic dose is about 20 µg / kg. In some embodiments, the effective therapeutic dose is about 30 µg / kg. In some embodiments, the effective therapeutic dose is about 40 µg / kg. In some embodiments, the effective therapeutic dose is about 45 µg / kg. In some embodiments, the effective therapeutic dose is about 50 µg / kg. In some embodiments, the effective therapeutic dose is about 60 µg / kg. In some embodiments, the effective therapeutic dose is about 100 µg / kg. In some embodiments, the effective therapeutic dose is about 100 µg / kg. In some embodiments, the effective therapeutic dose is about 130 µg / kg. In some embodiments, the effective therapeutic dose is about 150 µg / kg. In some embodiments, the effective therapeutic dose is about 200 µg / kg. In some embodiments, the effective therapeutic dose is about 300 µg / kg. In some embodiments, the effective therapeutic dose is about 400 µg / kg. In some embodiments, the effective therapeutic dose is about 450 µg / kg. In some embodiments, the dose is about 500 µg / kg. In some embodiments, the effective therapeutic dose is about 600 µg / kg. In some embodiments, the effective therapeutic dose is about 750 µg / kg. In some embodiments, the effective therapeutic dose is about 1000 µg / kg. In some embodiments, the effective therapeutic dose is about 1300 µg / kg. In some embodiments, the effective therapeutic dose is about 1500 µg / kg. In some embodiments, the effective therapeutic dose is about 2000 µg / kg. In some embodiments, the effective therapeutic dose is about 3000 µg / kg. In some embodiments, the effective therapeutic dose is about 4000 µg / kg. In some embodiments, the effective therapeutic dose is about 4500 µg / kg. In some embodiments, the effective therapeutic dose is about 6000 µg / kg.

[0463] In some embodiments, this disclosure provides kits and methods for treating diseases and disorders (e.g., blood disorders or disorders) in mammalian subjects requiring such treatment. In some embodiments, the blood disorder or disorder is acute myeloid leukemia (AML). In some embodiments, the AML is relapsed or refractory. In some embodiments, the blood disorder or disorder is blastic plasmacytoid dendritic cell tumor (BPDCN). In some embodiments, the blood disorder or disorder is B-cell acute lymphoblastic leukemia (B-ALL). In some embodiments, the blood disorder or disorder is high-risk myelodyplasia (HR-MDS). In some embodiments, the mammalian subject is a human patient. In some embodiments, the human patient is an adult patient. In some embodiments, the human patient is a pediatric patient.

[0464] The binding protein disclosed herein can be used in many different applications. For example, in one embodiment, the subject-binding protein can be used to reduce or eliminate cells carrying epitopes recognized by the binding domain of the binding protein. In another embodiment, the subject-binding protein is effective in reducing the concentration of circulating soluble antigens or eliminating circulating soluble antigens. In yet another embodiment, the subject-binding protein is effective as an NK cell binder (NKCE).

[0465] In another embodiment, the CD123 NKCE can be used to treat diseases or disorders associated with abnormal immune cells (e.g., bone marrow cells, B cells). In some embodiments, the abnormal immune cells (e.g., bone marrow cells or B cells) express CD123.

[0466] In some embodiments, the CD123 NKCE of this disclosure may be specifically used to treat diseases or disorders within the category of leukemia or myelodysplastic syndromes. In some embodiments, the leukemia is AML. In some embodiments, the disease or disorder is blastic plasmacytoid dendritic cell neoplasm (BPDCN). In some embodiments, the leukemia is B-ALL. In some embodiments, the myelodysplastic syndrome is high-risk myelodysplastic syndrome (HR-MDS).

[0467] Acute myeloid leukemia (AML)

[0468] Newly diagnosed, newly diagnosed AML patients eligible for intensive induction therapy typically receive induction therapy based on cytarabine and anthracyclines, followed by consolidation chemotherapy with cytarabine or other agents. Patients with AML are eligible to receive liposomal formulations of donomycin / cytarabine. With a better understanding of the molecular drivers of AML, newer molecularly targeted therapies have emerged. Elderly patients (>75 years) or those with comorbidities that preclude high-intensity chemotherapy induction are treated with less intensive therapies, including venetum / hypomethylating agent combinations, which offer better outcomes than previous monotherapy regimens.

[0469] Allogeneic stem cell transplantation is offered to patients with high-risk disease who have achieved first or subsequent remission, provided they have adequate organ function and a suitable source of stem cells. While approximately 15% of patients >60 years of age and 40% of patients <60 years of age who receive intensive induction chemotherapy for AML and / or allogeneic stem cell transplantation are cured, patients with relapsed or refractory (r / r) AML exhibit poorer outcomes after initial therapy. Although several new agents have been developed for r / r AML, it is generally incurable. Short duration of remission (e.g., <6 months), adverse genetic factors, prior allogeneic transplantation, older age, and general health status are factors associated with poorer survival outcomes in the context of r / r AML, partly because these factors limit eligibility for further intensive therapy.

[0470] The relative 5-year survival rate for patients with AML from 2011 to 2017 was 29.5%. In 2022, the U.S. is projected to see 20,050 new cases of AML, with a median age at diagnosis of 68 years. Recent clinical trial data indicate that the 3-year overall survival rate is only 50% to 60% among adults who have achieved complete remission. Despite advances in understanding the pathophysiology of AML and recognizing its molecular heterogeneity, developing viable treatments for patients with AML remains challenging.

[0471] BPDCN (Bermatoblastic Plasmacytic Dendritic Cell Tumor)

[0472] Blastoblastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive hematologic malignancy caused by plasmacytoid dendritic cell progeny. Several different nomenclatures were used to describe BPDCN in the past until the 2008 World Health Organization (WHO) classification described it as an entity under the family of acute myeloid leukemia (AML) and related tumors. In a subsequent WHO reclassification, it was given its own separate category under myeloid neoplasms, reflecting its unique pathobiology. BPDCN typically presents as cutaneous lesions with or without bone marrow involvement, lymphadenopathy, splenomegaly, cytopenia, and sometimes extramedullary involvement. Almost all cases are associated with CD123 overexpression on malignant cells.

[0473] BPDCN is suggested by biopsy of skin lesions, which may reveal medium-sized blast cells (i.e., immature cells) infiltrating the dermis but not the epidermis. These cells exhibit irregular nuclei, fine chromatin, and at least one small nucleolus. Such blast cells can also be observed in the circulation, bone marrow, or other tissues that may suggest BPDCN. However, diagnosis of this disease requires identification of the cell line plasmacytoid dendritic cell blast cells, rather than AML blast cells, T-cell lymphoblastic lymphoma blast cells, or aggressive NK-cell leukemia (NKL) blast cells.

[0474] Currently, no controlled studies have determined the optimal treatment for BPDCN. Studies of a small number of individuals with this disease have found that standard chemotherapy regimens for initial induction therapy in AML, ALL, and high-grade lymphoma yielded complete remission rates of 77%, 93%, and 80%, respectively, in pediatric PBDN, and 47%, 77%, and 53%, respectively, in adult PBDN. However, these remissions are transient: the median time from treatment to relapse or death is 12 months in children and 6.8 months in adults. Given these poor remission and survival rates, additional treatments have been added to the initial therapy regimen.

[0475] Currently, there is one approved treatment for BPDCN in the United States. This treatment (Tagraxofusp-erzs) is a fusion protein composed of IL-3 fused with diphtheria toxin. Other treatments (including CD123-specific CAR-T cells and venetumolat) are being investigated as potential treatments for BPDCN; however, more research is needed to find effective treatment options.

[0476] B-cell acute lymphoblastic leukemia and high-risk myelodystrophy

[0477] Similar to AML, the most effective treatments for B-ALL and HR-MDS include intensive multidrug chemotherapy and / or allogeneic stem cell transplantation; however, many patients with these conditions are elderly or have comorbidities that preclude such high-intensity, highly toxic therapies. Furthermore, not all patients are cured by intensive therapy and / or allogeneic transplantation, and there is a risk of treatment-related toxicities.

[0478] Potentially curative treatment for B-ALL requires high-intensity therapy that may be associated with significant toxicity. For B-ALL, a two-year course of intensive multi-drug chemotherapy and / or allogeneic stem cell transplantation can cure a significant proportion of younger patients (age < 40 years); however, outcomes are much worse in older patients. While salvage therapy with CD19-guided chimeric antigen receptor-T-cell (CAR-T) therapy (e.g., tisagenlecleucel) has resulted in durable remissions, it is not approved for patients over 25 years of age. Furthermore, CD123 is frequently expressed on B-ALL cells at both diagnosis and relapse, suggesting that CD123-guided therapies may be effective in this indication, even in the context of disease resistant to CD19-guided therapies such as tisagenlecleucel or bonnetumab (a T-cell conjugating antibody targeting CD18). For HR-MDS, hypomethylating agents have been the primary treatment, while other treatment options are limited. Allogeneic stem cell transplantation offers the only known cure for HR-MDS, but it is not always a viable treatment option given the patient's age and comorbidities.

[0479] IV. Pharmaceutical Compositions and Their Administration

[0480] The methods for preparing and administering the CD123 NKCE of this disclosure to subjects are well known or readily determined by those skilled in the art. The NKCE of this disclosure can be administered orally, parenterally, by inhalation, or topically. As used herein, parenterally includes intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all of the described forms of administration are explicitly contemplated within the scope of this disclosure, the form of administration will be a solution for injection, particularly a solution for intravenous or intra-arterial injection or infusion. Typically, suitable pharmaceutical compositions for injection may contain buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbate), stabilizers as needed (e.g., human albumin), etc. In some embodiments, the NKCE can be delivered directly to the site of the diseased cell population, thereby increasing the exposure of diseased tissue to the therapeutic agent.

[0481] Formulations intended for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffer media. In the compositions and methods disclosed herein, pharmaceutically acceptable carriers include, but are not limited to, 0.01–0.1 M (e.g., 0.05 M) phosphate buffer or 0.8% saline. Other common parenteral media include sodium phosphate solutions, Ringer's dextran, dextran and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous media include fluids and nutritional supplements, electrolyte supplements, such as those based on Ringer's dextran, etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases. More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (in the case of water solubility) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be a fluid available for easy injection. It should be stable under manufacturing and storage conditions and will typically be preserved against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings (e.g., lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0482] In many cases, isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride, will be included in the composition. Extended absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0483] In any case, a sterile injectable solution can be prepared by incorporating an active compound (e.g., NKCE itself or in combination with other active agents) in a desired amount into a suitable solvent, followed by filtration and sterilization, the solvent having, as needed, one or a combination of components listed herein. Typically, a dispersion is prepared by incorporating the active compound into a sterile medium containing a base dispersion medium and other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, exemplary preparation methods include vacuum drying and freeze-drying, which produce a powder of the active ingredient plus any other desired components from a previously sterile filtered solution. Injectable formulations are processed according to methods known in the art, filled into containers (such as ampoules, bags, bottles, syringes, or vials), and sealed under sterile conditions. Furthermore, formulations can be packaged and marketed as kits. Such articles will typically have labels or instructions for use indicating that the relevant composition may be used to treat subjects who have or are susceptible to autoimmune disorders or neoplastic disorders.

[0484] The effective dosage of the compositions of this disclosure for treating the aforementioned conditions varies depending on many different factors, including the method of administration, target site, patient's physiological state, whether the patient is human or animal, other pharmaceutical agents administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic mammals, can also be treated. The therapeutic dosage can be gradually adjusted using conventional methods known to those skilled in the art to optimize safety and efficacy.

[0485] The CD123 NKCE protein of this disclosure can be administered multiple times. The interval between single doses can be weekly, monthly, or annually. The interval can also be irregular, as indicated by measuring the blood levels of the patient's Fc domain variant or antigen. In some methods, the dose is adjusted to achieve a plasma concentration of about 1-1000 μg / ml of the modified binding peptide, and in others about 25-300 μg / ml. Alternatively, the Fc domain variant can be administered as a sustained-release formulation, in which case less frequent administration is required. For antibodies, the dose and frequency vary based on the antibody's half-life in the patient. Typically, humanized antibodies exhibit the longest half-life, followed by chimeric antibodies and non-human antibodies.

[0486] The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic use, a composition containing the polypeptides or mixtures thereof of the present invention is administered to a patient who is not yet in a disease state to enhance the patient's resistance. Such an amount is defined as the "preventive effective dose". In this use, the precise amount again depends on the patient's health status and general immunity, but typically ranges from about 0.001 to about 25 mg / kg / dose, especially from about 0.003 to about 6.0 mg / kg / dose. Relatively low doses are administered at relatively low intervals over long periods. Some patients continue treatment for the rest of their lives. In therapeutic use, it is sometimes necessary to administer relatively high doses (e.g., about 1 to 400 mg / kg antibody / dose, more commonly about 5 to 25 mg for radioimmunoconjugates, and more commonly higher doses for cytotoxic-drug modified antibodies) at relatively short intervals until disease progression is reduced or terminated, or until the patient shows partial or complete improvement in disease symptoms. Thereafter, a prophylactic regimen can be administered to the patient.

[0487] The NKCE of this disclosure can be administered, as needed, in combination with other agents that effectively treat the disorder or condition requiring treatment (e.g., prophylactic or therapeutic). The effective single-dose therapeutic dose (i.e., therapeutically effective dose) of the 90Y-labeled modified antibody of this disclosure ranges from about 5 to about 75 mCi, such as from about 10 to about 40 mCi. The effective single-dose non-bone marrow ablation dose of the 131I-modified antibody ranges from about 5 to about 70 mCi or from about 5 to about 40 mCi. The effective single-dose ablation dose (i.e., potentially requiring autologous bone marrow transplantation) of the 131I-labeled antibody ranges from about 30 to about 600 mCi, such as from about 50 to less than about 500 mCi. With the conjugation of chimeric antibodies, due to their longer circulating half-life relative to mouse antibodies, the effective single-dose non-bone marrow ablation dose of the iodine-131-labeled chimeric antibody ranges from about 5 to about 40 mCi, such as less than about 30 mCi. For example, the imaging standard for 111In markers is typically less than about 5 mCi.

[0488] While NKCEs can be administered as described above, it must be emphasized that in other embodiments, the peptides can be administered as a first-line therapy to otherwise healthy patients. In such embodiments, NKCEs can be administered to patients with normal or average red bone marrow reserves and / or patients who have not received and are currently not receiving treatment. As used herein, administration of the peptide in combination with adjuvant therapy or in combination means the sequential, simultaneous, concurrent, parallel, accompanying, or simultaneous administration or application of the therapy and the disclosed antibody. Those skilled in the art will understand that the administration or application of various components of a combination therapy regimen can be timed to enhance the overall effectiveness of the treatment.

[0489] As previously discussed, the NKCEs, their antibodies, therapeutic peptides, or NKCE fusion peptides disclosed herein can be administered in pharmaceutically effective amounts for in vivo treatment of mammalian disorders. In this regard, it will be understood that the disclosed NKCEs will be formulated to facilitate the administration of the active agent and to promote their stability.

[0490] The pharmaceutical compositions according to this disclosure may comprise a pharmaceutically acceptable, non-toxic, sterile carrier, such as physiological saline, non-toxic buffer, preservative, etc. For the purposes of this application, a pharmaceutically effective amount of CD123 NKCE, conjugated or unconjugated with a therapeutic agent, should be considered to be an amount sufficient to achieve effective binding to the antigen and to provide a benefit (e.g., improvement of symptoms of a disease or disorder or detection of a substance or cell). In the case of plasma cells, the polypeptide can interact with selected antigens on immune-responsive cells and provide an increase in the death of those cells. Of course, the pharmaceutical compositions of this disclosure can be administered in a single dose or multiple doses to provide a pharmaceutically effective amount of the modified conjugated polypeptide.

[0491] Consistent with the scope of this disclosure, the CD123 NKCE of this disclosure can be administered to humans or other animals in an amount sufficient to produce a therapeutic or preventative effect, according to the aforementioned treatment methods. The CD123 NKCE of this disclosure can be administered to such humans or other animals in conventional dosage forms (prepared by combining the antibody of this disclosure with a conventionally pharmaceutically acceptable carrier or diluent according to known techniques). Those skilled in the art will recognize that the form and characteristics of a pharmaceutically acceptable carrier or diluent are determined by the amount of the active ingredient combined with it, the route of administration, and other well-known variables. Those skilled in the art will further understand that mixtures comprising one or more of the binding peptides described in this disclosure can prove particularly effective.

[0492] The contents of any articles, patents, and patent applications mentioned or cited herein, as well as all other documents and electronically available information, are hereby incorporated in their entirety by reference, to the extent that each individual publication expressly and individually indicates its inclusion by reference. The applicant reserves the right to actually incorporate any and all material and information from any such articles, patents, patent applications, or other physical and electronic documents into this application.

[0493] While the invention has been described with reference to specific embodiments thereof, those skilled in the art will understand that various changes and equivalent substitutions can be made without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations can be made to the methods described herein using appropriate equivalents without departing from the scope of the embodiments disclosed herein. Furthermore, many modifications can be made to adapt specific circumstances, materials, composition, methods, or one or more method steps to the purpose, spirit, and scope of the invention. All such modifications are intended to fall within the scope of the appended claims. Certain embodiments have now been described in detail and will be more clearly understood by referring to the following embodiments, which are included for illustrative purposes only and are not intended to be restrictive.

[0494] sequence list

[0495] In the protein sequence symbols used in this article, according to standard usage and convention, the left-hand direction is the direction of the amino terminus ("N-terminus"), and the right-hand direction is the direction of the carboxyl terminus ("C-terminus").

[0496] SEQ Name Sequence 1 V H1 CDR-H1 anti-CD123 CD123-1]]> GYSFTDYYMK 2 V H1 CDR-H2 anti-CD123 CD123-1 DIIPSSGATF 3 V H1 CDR-H3 anti-CD123 CD123-1 SHLLRASWFAY 4 Anti-CD123 CD123-2 GFTFSHYN 5 V H1 CDR-H2 anti-CD123 CD123-2 ITYDDHST 6 V H1 CDR-H3 anti-CD123 CD123-2 ARLVNYAFAY 7 V L1 CDR-L1 anti-CD123 CD123-1]]> ESSQSLLSSGNQKNYLT 8 V L1 CDR-L2 anti-CD123 CD123-1]]> WASTRES 9 V L1 CDR-L3 anti-CD123 CD123-1]]> QNDYSYPYT 10 V L1 CDR-L1 anti-CD123 CD123-2 QTVGNN 11 V L1 CDR-L2 anti-CD123 CD123-2 YAS 12 V L1 CDR-L3 anti-CD123 CD123-2]]> QRMYNSPT 13 V H2 CDR-H1 anti-NKp46 NKp46-1 DYVIN 14 V H2 CDR-H2 anti-NKp46 3D9 and NKp46-1 EIYPGSGTNYYNEKFKA 15 V H2 CDR-H3 anti-NKp46 3D9 and NKp46-1]]> 16 V H2 CDR-H1 anti-NKp46 NKp46-213G4 RGRYGLYAMDY 17 <![CDATA[V H2 CDR-H2 Anti-NKp46 NKp46-2 SDYAWN 18 <![CDATA[V H2 CDR-H3 Anti-NKp46 NKp46-2 YITYSGSTSYNPSLES 19 <![CDATA[V H2 CDR-H1 Anti-NKp46 NKp46-3 GGYYGSSWGVFAY 20 <![CDATA[V H2 CDR-H2 Anti-NKp46 NKp46-3 EYTMH 21 <![CDATA[V H2 CDR-H3 Anti-NKp46 NKp46-3 GISPNIGGTSYNQKFKG 22 <![CDATA[V H2 CDR-H1 Anti-NKp46 NKp46-4 RGGSFDY 23 <![CDATA[V H2 CDR-H2 Anti-NKp46 NKp46-4 SFTMH 24 <![CDATA[V H2 CDR-H3 Anti-NKp46 NKp46-4 YINPSSGYTEYNQKFKD 25 <![CDATA[V H2 CDR-H2 Anti-NKp4613G4 GSSRGFDY 26 <![CDATA[V H2 CDR-H3 Anti-NKp4613G4 YITYSGSTNYNPSLKS 27 <![CDATA[V L2 CDR-H1 is effective against NKp463D9 and NKp46-1. CWDYALYAMDC 28 <![CDATA[V L2 CDR-H2 is effective against NKp463D9 and NKp46-1. RASQDISNYLN 29 <![CDATA[V L2 CDR-H3 is effective against NKp463D9 and NKp46-1. YTSRLHS 30 <![CDATA[V L2 CDR-H1 Anti-NKp46 NKp46-2 QQGNTRPWT 31 <![CDATA[V L2 CDR-H2 Anti-NKp46 NKp46-213G4 RVSENIYSYLA 32 <![CDATA[V L2 CDR-H3 Anti-NKp46 NKp46-2 NAKTLAE 33 <![CDATA[V L2 CDR-H1 Anti-NKp46 NKp46-3 QHHYGTPWT 34 <![CDATA[V L2 CDR-H2 Anti-NKp46 NKp46-3 RASQSISDYLH 35 <![CDATA[V L2 CDR-H3 Anti-NKp46 NKp46-3 YASQSIS 36 <![CDATA[V L2 CDR-H1 Anti-NKp46 NKp46-4 QNGHSFPLT 37 <![CDATA[V L2 CDR-H2 Anti-NKp46 NKp46-4 RASENIYSNLA 38 <![CDATA[V L2 CDR-H3 Anti-NKp46 NKp46-4 AATNLAD 39 <![CDATA[V L2 CDR-H2 Anti-NKp4613G4 QHFWGTPRT 40 <![CDATA[V L2 CDR-H3 Anti-NKp4613G4 RTSENIYSYLA 41 QHHYDTPLT VH-full-length-anti-CD123 42 EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYMKWARQMPGKGLEWMGDIIPSSGATFYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWFAYWGQGTMVTVSS VH-full-length-anti-CD123 43 EVQLVESGGGLVQPGRSLKLSCAASGFTFSHYNMAWVRQAPKKGLEWVATITYDDHSTYYRDSVKGRFTISRDTAKSTLYLQMDSLRSEDTATYYCARLVNYAFAYWGQGTLVTVSS VL-full-length-anti-CD123 44 DIVMTQSPDSLAVSLGERATINCESSQSLLSSGNQKNYLTWYQQKPGQPPKPLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIK VL-full-length-anti-CD123 45 NIVMTQSPKSMSISVGDRVTMNCKASQTVGNNIAWYQQKPGLSPQLLIDYASNRYTGVPNRFTGGGYGTDFILTINSVQAEDAAFYYCQRMYNSPTFGGGTKLELK VH-full-length-anti-NKp46 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYVINWVRQAPGQGLEWMGEIYPGSGTNYYNEKFKAKATITADKSTSTAYMELSSLRSEDTAVYYCARRGRYGLYAMDYWGQGTTVTVSS 46 VH-Full-length-Anti-NKp46 NKp46-1 QVQLQQSGPELVKPGASVKMSCKASGYTFTDYVINWGKQRSGQGLEWIGEIYPGSGTNYYNEKFKAKATLTADKSSNIAYMQLSSLTSEDSAVYFCARRGRYGLYAMDYWGQGTSVTVSS 47 VH-Full-length-Anti-NKp46 NKp46-2 EVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYITYSGSTSYNPSLESRISITRDTSTNQFFLQLNSVTTEDTATYYCARGGYYGSSWGVFAYWGQGTLVTVSA 48 VH-Full-length-Anti-NKp46 NKp46-3 EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGISPNIGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRGGSFDYWGQGTTLTVSS 49 VH-Full-length-Anti-NKp46 NKp46-4 QVQLQQSAVELARPGASVKMSCKASGYTFTSFTMHWVKQRPGQGLEWIGYINPSSGYTEYNQKFKDKTTLTADKSSSTAYMQLDSLTSDDSAVYYCVRGSSRGFDYWGQGTLVTVSA 50 VH-Full-length-Anti-NKp46 10B8 QVQLQESGPGLVKPSQTLSLTCTVSGYSISSDYAWNWIRQPPGKGLEWIGYITYSGSTSYNPSLESRVTISRDTSKNQFSLKLSSVTAADTAVYYCARGGYYGSSWGVFAYWGQGTLVTVSS 51 VH-Full-length-Anti-NKp46 12E12 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSFTMHWVRQAPGQGLEWIGYINPSSGYTEYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCVRGSSRGFDYWGQGTLVTVSS 52 VH-Full-length-Anti-NKp46 13G4 QVQLQESGPGLVKPSQTLSLTCTVSGYSISSDYAWNWIRQPPGKGLEWIGYITYSGSTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARCWDYALYAMDCWGQGTTVTVSS 53 VL-full-length-anti-NKp46 3D9 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQQGNTRPWTFGGGTKVEIK 54 VL-full-length-anti-NKp46 NKp46-1 DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTINNLEQEDIATYFCQQGNTRPWTFGGGTKLEIK 55 VL-full-length-anti-NKp46 NKp46-2 DIQMTQSPASLSASVGETVTITCRVSENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPWTFGGGTKLEIK 56 VL-full-length-anti-NKp46 NKp46-3 DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELK 57 <![CDATA[VL - Full length - Anti-NKp46 NKp46-4]]> DIQMIQSPASLSVSVGETVTITCRASENIYSNLAWFQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGIYYCQHFWGTPRTFGGGTKLEIK 58 VL-full-length-anti-NKp46 10B8 DIQMTQSPSSLSASVGDRVTITCRVSENIYSYLAWYQQKPGKAPKLLVYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPWTFGGGTKVEIK 59 VL-full-length-anti-NKp46 12E12 DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWFQQKPGKAPKLLVYAATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPRTFGGGTKVEIK 60 VL-full-length-anti-NKp46 13G4 DIQMTQSPSSLSASVGDRVTITCRTSENIYSYLAWCQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYDTPLTFGQGTKLEIK 61 Polypeptide chain (I) MSVPTQVLGLLLLWLTDARCDIVMTQSPDSLAVSLGERATINCESSQSLLSSGNQKNYLTWYQQKPGQPPKPLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 62 Polypeptide chain (II) MEWSWVFLFFLSVTTGVHSEVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYMKWARQMPGKGLEWMGDIIPSSGATFYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWFAYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSTGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYVINWVRQAPGQGLEWMGEIYPGSGTNYYNEKFKAKATITADKSTSTAYMELSSLRSEDTAVYYCARRGRYGLYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHS 63 Polypeptide chain (III) MSVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQQGNTRPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 64 Cleaved F25 polypeptide chain (I) (without leader peptide) DIVMTQSPDSLAVSLGERATINCESSQSLLSSGNQKNYLTWYQQKPGQPPKPLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 65 Cleaved F25 polypeptide chain (II) (without leader peptide) EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYMKWARQMPGKGLEWMGDIIPSSGATFYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWFAYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSTGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYVINWVRQAPGQGLEWMGEIYPGSGTNYYNEKFKAKATITADKSTSTAYMELSSLRSEDTAVYYCARRGRYGLYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHS 66 Cleaved F25 polypeptide chain (III) (without leader peptide) DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQQGNTRPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 67 Cκ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 68 <![CDATA[C H 1]]> ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV 69 <![CDATA[(C H 2-C H 3) A ]]> APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 70 <![CDATA[(C H 2-C H 3) B ]]> APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 71 <![CDATA[C H 2]]> APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK 72 <![CDATA[(C H 2-C H 3) A C H 3]]> GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK 73 <![CDATA[(C H 2-C H 3) B The C-terminus truncated H 3]]> GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 74 <![CDATA[Hinge 1]]> DKTHTCPPCP 75 <![CDATA[Hinge 2]]> EPKSCDKTHTCPPCP 76 <![CDATA[Connector 1]]> STGS 77 <![CDATA[Hinge 3]]> EPKSCDKTHS 78 <![CDATA[hinge ALT1 > EPKSCDKTH 79 <![CDATA[hinge ALT2 > EPKSCDKTHT 80 Human NKp46 extracellular domain (ECD) MSSTLPALLCVGLCLSQRISAQQQTLPKPFIWAEPHFMVPKEKQVTICCQGNYGAVEYQLHFEGSLFAVDRPKPPERINKVKFYIPDMNSRMAGQYSCIYRVGELWSEPSNLLDLVVTEMYDTPTLSVHPGPEVISGEKVTFYCRLDTATSMFLLLKEGRSSHVQRGYGKVQAEFPLGPVTTAHRGTYRCFGSYNNHAWSFPSEPVKLLVTGDIENTSLAPEDPTFPADTWGTYLLTTETGLQKDHALWDHTAQN 81 Macaca fascicularis NKp46-FlagM2 extracellular domain (ECD) MSSTLRALLCLGLCLSQRISAPKQTLPKPIIRAESTYMVPKEKQATLCCQGSYGAVEYQLHFEGSLFAVERPKPPERINGVKFHIPDMNSRKAGRYSCIYRVGELWSERSDLLDLVVTEMYDTPTLSVHPGPEVTSGEKVTFYCRLDTATSMFLLLKEGRSRDVQRSYGKVQAEFPMGPVTTAHRGSYRCFGSYNNYAWSFPSEPVKLLVTGDIENTSLAPTDPTFPDSWDTCLLTRETGLQKDLALWDHTAQNDYKDDDDK 82 Human CD123 extracellular domain (ECD) TKEDPNPPITNLRMKAKAQQLTWDLNRNVTDIECVKDADYSMPAVNNSYCQFGAISLCEVTNYTVRVANPPFSTWILFPENSGKPWAGAENLTCWIHDVDFLSCSWAVGPGAPADVQYDLYLNVANRRQQYECLHYKTDAQGTRIGCRFDDISRLSSGSQSSHILVRGRSAAFGIPCTDKFVVFSQIEILTPPNMTAKCNKTHSFMHWKMRSHFNRKFRYELQIQKRMQPVITEQVRDRTSFQLLNPGTYTVQIRARERVYEFLSAWSTPQRFECDQEEGANTRAWR 83 Human Fc-γ receptor IIIA (CD16A) MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK 84 Human Fc-γ receptor 3A (CD16A) (V176F polymorphic variant) MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK 85 NKp46-IC_F25 fragment 1 EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLFSDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYRYSPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 86 NKp46-IC_F25 Fragment 2 EVQLVESGGGLVQPGRSLRLSCAASGFTFDNYAMHWVRQAPGKGLEWVSGISRSSGDIDYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARGGVGSFDTWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSTGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYVINWVRQAPGQGLEWMGEIYPGSGTNYYNEKFKAKATITADKSTSTAYMELSSLRSEDTAVYYCARRGRYGLYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHS 87 NKp46-IC_F25 Fragment 3 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQQGNTRPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 88 ​ DIVMTQSPDSLAVSLGERATINCESSQSLLSSGNQKNYLTWYQQKPGQPPKPLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 89 moNKp46-huCD123_Fragment 2 EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYMKWARQMPGKGLEWMGDIIPSSGATFYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWFAYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSTGSEVQLVESGGGLVKPGGSLKLSCAASGFTFSDYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARGTTIFNYFEYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHS 90 moNKp46-huCD123_Fragment 3 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYEIPPTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 91 NKp46-CD123_Fragment 1 DIVMTQSPDSLAVSLGERATINCESSQSLLSSGNQKNYLTWYQQKPGQPPKPLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 92 NKp46-CD123_F5 fragment 2 EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYMKWARQMPGKGLEWMGDIIPSSGATFYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWFAYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSTGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYVINWVRQAPGQGLEWMGEIYPGSGTNYYNEKFKAKATITADKSTSTAYMELSSLRSEDTAVYYCARRGRYGLYAMDYWGQGTTVTVSSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 93 NKp46-CD123_F5 Fragment 3 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQQGNTRPWTFGGGTKVEIKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHS 94 IC-CD123_F5 Fragment 1 DIVMTQSPDSLAVSLGERATINCESSQSLLSSGNQKNYLTWYQQKPGQPPKPLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 95 IC-CD123_F5 fragment 2 EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYMKWARQMPGKGLEWMGDIIPSSGATFYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWFAYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSTGSEVQLVQSGAEVKKSGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIFYPGDSSTRYSPSFQGQVTISADKSVNTAYLQWSSLKASDTAMYYCARRRNWGNAFDIWGQGTMVTVSSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 96 IC-CD123_F5 Fragment 3 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSTWTFGQGTKVEIKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHS 97 IC-hIGg1-ADCC-enhanced light chain EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLFSDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYRYSPRTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 98 IC-hIGg1-ADCC-enh heavy chain EVQLVESGGGLVQPGRSLRLSCAASGFTFDNYAMHWVRQAPGKGLEWVSGISRSSGDIDYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARGGVGSFDTWGQGT MVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0497] Example

[0498] The present invention is further illustrated by the following embodiments, which should not be construed as further limitations.

[0499] Example 1. In vitro binding of CD123 NKCE to human CD123 and NKp46 and tumor killing effect of CD123 NKCE

[0500] CD123 NKCE exhibits high affinity for human CD123 and NKp46 targets that bind through its variable region, with sub-nanomolar affinity for CD123 and nanomolar affinity for NKp46. CD123 NKCE also binds to human CD16a (FcγRIIIa; and other Fcγ receptors) through its wild-type active Fc domain, as shown in Table 2.

[0501] Table 2. Affinity of CD123 NKCE to human CD123, NKp46 and CD16a targets.

[0502]

[0503] Abbreviation: K D : Equilibrium dissociation constant; SD: Standard deviation; V / F: Valine / Phenylalanine

[0504] The binding of CD123 NKCE to its targets was confirmed at the cellular level using flow cytometry, indicating binding to cell surface CD123 on the human MOLM-13 AML cell line and to cell surface NKp46 and CD16a on human NK cells purified from healthy donors. CD123 NKCE co-conjugates NK cells, leading to tumor cell killing, by binding to NKp46 and CD16a and to CD123 on CD123-positive tumor cells. CD123 NKCE resulted in potent cytotoxic activity against MOLM-13 AML cells (approximately 20,000 CD123 receptors / cell). As expected, the cytotoxic activity of CD123 NKCE varied with the effector cell to target cell (E:T) ratio. Using KG-1a (approximately 3,000 sites / cell) and NB-4 (approximately 1,800 sites / cell), CD123 NKCE also resulted in specific cytotoxic activity against human AML cell lines expressing low CD123 antigen densities (ranging from 1,800 to 3,000 antigens / cell) (Table 3).

[0505] Table 3. Cytotoxic activity of fresh NK cells against AML cell lines exhibiting different CD123 receptor densities.

[0506]

[0507] CD123 NKCE co-conjugation against MOLM-13 AML cells and CD123-positive tumor cells led to NK cell activation, degranulation, and expression of effector molecules. CD123 NKCE mediated significant NK cell activation in the presence of MOLM-13 target cells, inducing the expression of CD69, CD107, TNFα, IFNγ, and MIP1β in a concentration-dependent manner. No NK cell activation was observed in the absence of target cells, thus validating an immune synapse-driven activation mechanism.

[0508] CD123 NKCE also exhibits in vitro activity against primary NK cells from AML patients, resulting in (i) concentration-dependent activation of primary NK cells in autologous AML blast cells (CD107 degranulation marker) (using primary blood samples from AML patients at diagnosis) and (ii) potentiogenic activity against AML blast cells (using healthy donor NK cells as effectors), relative to half-maximum effective concentration (EC50). 50 The concentration of rel was 3.5 ng / mL (23.2 pM).

[0509] Example 2. Development of in vitro and in vivo PK / PD models.

[0510] principle

[0511] Advances in immuno-oncology are at the forefront of transformative cancer treatment, focusing on the specific recognition and elimination of tumor cells by the host immune system. Several targeted immunotherapies targeting antigens frequently expressed on AML blasts (such as CD123), including bispecific T-cell binders (TCEs) and natural killer (NK) cell binders (NKCEs), are currently being clinically evaluated in patients with acute myeloid leukemia (AML). NK cell-based therapies represent a promising alternative to T-cell therapies for AML, without common adverse events such as cytokine release syndrome (CRS) or neurotoxicity. Specifically, NK cell binders are engineered, multifunctional antibody-like molecules that co-bind to tumor cells and NK cells by binding to tumor-specific antigens and NK activation receptors to trigger NK cell activation and tumor cell destruction.

[0512] A trifunctional NKCE targeting the CD123 tumor antigen has recently been described. The CD123-targeting NKCE also co-binds to NKp46 and CD16a (FcγRIIIa) (two major activating receptors on NK cells) and has demonstrated potent antitumor activity against primary AML blast cells. In preclinical studies, it has been shown to promote strong NK cell activation and cytokine secretion only in the presence of AML blast cells. The mechanism of action (MoA) of NKCE is comparable to that of TCE molecules, where NKCE brings tumor cells and NK cells closer together to trigger immune cell killing of tumor cells. Like TCE molecules, NKCE drives immune synapse formation, resulting in a bell-shaped concentration-response curve, where the width of the curve reflects the efficacy window and depends on factors controlling synapse formation (i.e., effector cell to tumor cell [E:T] ratio, target receptor expression, and binding affinity).

[0513] Several in vitro and in vivo PK / PD models have been developed for multispecific TCE or chimeric antigen receptor T-cell (CAR-T) therapies; however, a model-based transformation PK / PD approach targeting the NKCE molecule has not yet been established. Therefore, there is a need to develop synapse-driven PK / PD models targeting the trifunctional CD123-NKCE molecule for the transformation AML mechanism, using in vitro cytotoxicity and in vivo non-human primate (NHP) data.

[0514] method

[0515] Construction of preclinical datasets for model development

[0516] In vitro cytotoxicity assay

[0517] The efficacy of resting NK cells from two donors was evaluated at eight drug concentrations (1.5E-3, 1.5E-2, 1.5E-1, 1.5, 1.5E1, 1.5E2, 1.5E3, and 1.5E4 ng / mL) at different E:T ratios (10:1, 5:1, 1:1, and 1:5) for three incubation periods (1, 4, and 20 hours). 51 Cytotoxicity of Cr-stained MOLM-13 cells.

[0518] In vivo NHP study

[0519] In vivo NHP PK / PD models were developed using data from preclinical NHP PK / PD studies in monkeys following single-dose administration and repeated-dose administration (dose range: 0.5 to 3000 µg / kg via intravenous (IV) administration and 3.6 to 1000 µg / kg via subcutaneous administration) and in vitro PK / PD models.

[0520] In vitro PK / PD model for transformation of synaptic-related parameters

[0521] As described in this paper, an in vitro PK / PD model was developed. This model estimates synaptic-related PD parameters of cytotoxicity (i.e., synaptic potency [ECG]). 50 ], the maximum CD123+ cell killing rate constant at the synapse [k max [and Hill coefficient [γ]], assuming stable NK cell kinetics (not changing over time) and that no internalization occurred in the in vitro system. The concentrations of CD123-NKCE (B0), cell numbers (EF0 and TC0), NKp46 and CD123 receptor densities (RTdT and RTdE), and binding affinity (KD1, KD2, k off1 and k off2 The relevant parameters are fixed using values ​​obtained from internal company sources or literature.

[0522] In vivo NHP PK / PD model

[0523] An extended in vitro PK / PD model was used for in vivo NHP PK / PD modeling. In this in vivo model, in addition to fixed values ​​of synapse-related PD parameters, the following additional parameters were included: plasma PK (bioavailability [F], absorption rate constant [Ka], clearance rate [CL], intercompartmental clearance between the central and peripheral compartments [Q], distribution volume in the central compartment [V1], distribution volume in the peripheral compartment [V2]), and internalization of the trifunctional antibody-CD123 binary complex (k...). int Synapse-driven NKp46+ NK cell redistribution (Slp) NKThe effect of anti-drug antibodies (ADA) on PK (the rate constant of drug elimination caused by ADA [K]). ADA ] and the typical time until the emergence of ADA [T ADA ]) and tolerance feedback to CD123+ cell regeneration (k tol And δ). Furthermore, it is hypothesized that the trifunctional antibody spontaneously binds to CD123 expressed in compartments other than blood, leading to the formation of a binary complex of the trifunctional antibody and CD123 undergoing internalization. This additional conversion of CD123 expression is facilitated by k in TP and k out TP parameters were used to describe the data. PK and PD data from 23 of the 53 NHPs evaluated in the NHP study were included in the NHP PK / PD modeling. The remaining 30 NHPs received CD123 NKCEs at 100 and 3000 µg / kg (intravenous, twice weekly (biw)) and 3000 µg / kg (subcutaneous, biw), with PK data included for model development and PD data used for external validation. Observed baseline cell parameters (EF0 and TC0) were mapped as regression variables in the model. Data on censored PK and total CD123+ cells below the limit of quantification (BLQ) were processed using the M3 method (Bergstrand, M. and MO Karlsson, Handling data below the limit of quantification in mixed-effect models. AAPS J, 2009. 11(2): 371-80).

[0524] AML to PK / PD model

[0525] By assuming a model structure similar to NHP (with a few exceptions), the following is an example of converting AML patients into an in vivo NHPPK / PD model: using the maximum load of CD123+ cells (TC) max This replaces the tolerance feedback to tumor growth, and PK allometric scaling with exponents of 0.81, 0.57, 1.04, and 1.07 is applied to CL, Q, V1, and V2, respectively. System-specific and binding affinity-related parameters are adapted from known values ​​in patients or healthy subjects (e.g., EF0, TC0, TC). max NK cell turnover rate constant [k] out CD123+ cell proliferation constant [k] g [ ], RTdE, RTdT, KD1, and KD2. In contrast, biologically specific parameters unknown in patients (e.g., kin TP, α, and β) or drug-specific parameters (e.g., k) for which no explicit scaling method is used. int Slp NK k max EC 50 γ) were assumed to be the same as the parameters for NHP. Notably, the effect of ADA on CD123-NKCE clearance was not included in the AML PK / PD model because ADA is unlikely to be present in humans due to the use of a humanized CD123-NKCE construct.

[0526] Starting dose prediction

[0527] Simulations were performed to predict the initial intravenous dose of CD123-NKCE that would meet the following two criteria: i) the maximum concentration at the end of a single infusion (C eoi The dose prediction method assumes that the intensity of cytokine release syndrome (a rapid adverse reaction) will be lower than the minimum concentration (1.7 nmol / L) found to induce mild cytokine release in whole blood cytokine release assays; and that the CD123+ cell count will decrease by >95% at the end of the first treatment cycle (i.e., 28 days) following biw administration. This dose prediction method assumes that the intensity of cytokine release syndrome (a rapid adverse reaction) depends on the maximum concentration (C) achieved after a single dose of the biologic. max ).

[0528] Sensitivity analysis

[0529] To evaluate the uncertainty of the model related to efficacy at the predicted starting dose, GSA (A. Saltelli, S. Tarantola and KP-S. Chan (1999) A Quantitative Model-Independent Method for Global Sensitivity Analysis of Model Output), Technometrics, 41:1, 39-56, DOI: 10.1080 / 00401706.1999.10485594) and LSA were performed using the extended Fourier amplitude sensitivity test (eFAST). This allowed for estimation of the uncertainty of the model prediction of PK and the percentage change in CD123+ cell count relative to baseline (CFB%) at the selected FIH starting dose.

[0530] result

[0531] Characterization of in vitro data

[0532] The observed in vitro cytotoxicity data of MOLM-13 cells after co-incubation with CD123-NKCE at different concentrations, incubation time intervals, and different E:T ratios were fitted using an in vitro PK / PD model framework (Figure 4A). The model was used by fixing k with known values. g PD parameters were estimated using CD123 receptor density (RTdT) (Table 2).

[0533] Table 4. Summary of parameters fixed or estimated for the development of preclinical (in vitro and in vivo) PK / PD models and translational clinical PK / PD models targeting CD123 NKCE.

[0534]

[0535]

[0536]

[0537] Abbreviations: ADA, Anti-drug antibody; AML, Acute myeloid leukemia; CD, Differentiation cluster; IIV, Inter-individual variability; NHP, Non-human primate; NK, Natural killer; PD, Pharmacodynamics; PK, Pharmacokinetics; RSE, Relative standard error. a Standard deviation of IIV random effects; b Source: Actual concentration as a regression measure; c Source: Internal data; d Source: Baroni, ML et al., 41BB-based and CD28-based CD123-redirected T-cells ablate humannormal hematopoiesis in vivo. J Immunother Cancer, 2020. 8(1).; e Source: Estimated; f Source: Estimated from in vitro PK / PD models; g Source: Doubling time is approximately 50 hours; h Source: Based on a 24-hour doubling time; i Source: Calibrated; j Source: Stringaris, K., Orphan NKs! The mystery of the self-renewing NK cells. Blood, 2017. 129(14): pp. 1890-1891. k Source: Internal measurements of the human body; l Source: Scaling from NHP data;m Source: Estimated from NHP data; n Source: Jiang, o Rogitt model of random effects of IIIV; p Assume it is 95% higher than the baseline.

[0538] Model fitting showed good data fit at different incubation time points, except that the predictions were slightly overestimated for data with E:T ratios of 1:1 and 1:5 after 20 h of incubation (Figure 5). This can be attributed to the affected data. 51 The Cr was explained by spontaneous release from radiolabeled MOLM-13 cells, and the raw values ​​in the affected data were negative and needed to be normalized using control measurements before they could be used for modeling.

[0539] Characterization of in vivo PK / PD data of CD123 NKCE in NHP

[0540] Given the large number of target-mediated drug disposition (TMDD) and ADA observed in NHP, an in vivo NHP PK / PD model was developed in a sequential manner, starting with fitting data from intravenous and subcutaneous CD123-NKCE administration at time points < 1 week that showed linear PK without ADA effects, to ensure that linear PK parameters (CL, Q, V1, V2, F, and ka) were estimated with high accuracy (Table 1).

[0541] Remaining time points (i.e., ≥ 1 week) from data on intravenous and subcutaneous administration at 3000 µg / kg were included in the model to characterize the ADA effect. The model-estimated T... ADA The estimated time was approximately 2 weeks (Table 1), consistent with the observed start time of ADA (Figure 6). K was estimated. ADA It is approximately 400 times the rate constant of nonspecific elimination. (1.94 1 / h vs. 0.005 1 / h). The high inter-individual variability (IIV) observed in NHP PK at time points > 2 weeks (Figure 7A) can be explained by the heterogeneity of NHP response to human trifunctional antibody administration (Table 1).

[0542] After incorporating all PK data used for modeling, the model needs to account for the binding of CD123-NKCEs to additional CD123 receptors expressed in compartments other than blood to characterize TMDD. Without this assumption, the model was observed to consistently over-predict the PK profile at doses exhibiting TMDD (data not shown). Therefore, an additional turnover model was included to characterize this additional CD123 expression under this assumption. The baseline CD123+ cell count observed in blood was directly correlated with the baseline additional CD123 expressed elsewhere, depicted by scaling functions parameterized as α and β.

[0543] CD123+ cell count curves alone showed that early rebound of CD123+ cell depletion was generally associated with early ADA production. Of all the animals exhibiting a rebound at approximately 2 weeks, one animal had the lowest ADA and showed prolonged CD123+ cell depletion compared to other NHPs exhibiting high CD123-NKCE clearance in the presence of ADA (Figure 8). Therefore, characterizing the effect of ADA alone on PK is important before models can adequately characterize the CD123+ cell profile. CD123+ cell count data during the rebound phase showed a satisfactory fit in the visual predictive test (VPC) (Figure 6B).

[0544] Because the study focused on healthy NHP, the CD123+ cell rebound was ultimately offset by tolerance feedback mechanisms, thus returning to baseline readings. Therefore, a regulator-mediated tolerance model was incorporated to act on parameter k. g Based on external validation using biw data, the model adequately predicted CD123+ cell depletion, but underpredicted data during the rebound phase, even after excluding extremely high cell counts (>200 cells / µL) (Figure 9).

[0545] The decrease in NKp46+ NK cells observed in the blood after CD123-NKCE administration can be explained by synapse-driven NK cell redistribution. The NKp46+ NK cell turnover model includes the effect of synaptic concentration on NK cell turnover. out The linear slope function of the stimulus (Fig. 4B) shows a good fit to the observations (Fig. 6C). According to estimates, NKp46+ NK cell redistribution increases at a rate of 5.72 1 / (mol / L) with increasing synaptic concentration (Table 1).

[0546] Overall, the model is satisfactory (Figures 6A-6C), and the parameters are estimated with good accuracy (Table 1).

[0547] Transform AML PK / PD model

[0548] The linear PK parameters CL, Q, V1, and V2 for allometric scaling of CD123-NKCE from NHP to humans were 0.18 mL / h / kg, 0.37 mL / h / kg, 0.042 L / kg, and 0.031 L / kg, respectively (Table 1).

[0549] The following inputs were applied to the AML model converted from the NHP model: 1) Lower EF0 and higher TC0 counts, resulting in a much lower E:T ratio; 2) Lower RTdT value; 3) Binding affinity (KD1 and KD2) values ​​obtained from in vitro experiments using human cells; 4) Recalculated k based on the 24-hour doubling time. g ;5) k calculated based on a 14-hour half-life out ; and 6) TC in CD123+ cells in the blood max The assumption is that the value is 95% higher than the baseline.

[0550] Uncertainty in predicting safety and efficacy at the starting dose

[0551] Of all the parameters examined using GSA, the one controlling CD123+ cell growth (k) was shown to be the most important. g Tumor killing through synapse formation (k) max EC 50 and γ), the internalization rate constant (k) in the case of CD123 receptor. int Other CD123 expression (α, β, and k) in TP), baseline count of CD123+ cells (TA0), and total TC of CD123+ cells max The parameters significantly affect the prediction of tumor cell killing (eFAST full-order index > 0.05; Figure 10A).

[0552] According to the LSA results, k g and k max The most sensitive parameters are α, γ, and k. in TP, k int and EC 50 The most sensitive parameter is k, followed by others that are less sensitive (Figure 6B). These results largely agree with the full-order exponent ranking of the GSA, as depicted in Figure 6A. It is noteworthy that k... g Increase by more than 2 times or k max A reduction of >2-fold will result in unsustainable CD123+ cell depletion >95% throughout the first treatment cycle (Figure 10B). For α, γ, and k... in TP, k int and EC 50Increasing the parameter value will primarily affect CD123 depletion during the rebound period that occurs 21 days after the first CD123-NKCE dose (Figure 10B).

[0553] Simulation of initial dose prediction

[0554] Simulations were performed to predict the PK and %CFB (changes relative to baseline) of CD123+ cell counts after biw administration of CD123-NKCE. A fixed dose of at least 100 µg / kg was required during the first dosing cycle to achieve a >95% reduction in CD123+ cell counts (Figure 7A). However, this first dose of 100 µg / kg may induce cytokine release, as predicted C… eoi This exceeded the lowest concentration level of CD123-NKCE observed for cytokine release from in vitro whole blood assays (1.7 nmol / L) (Figure 7B). To mitigate the risk of any adverse events, such as infusion-related cytokine release, we simulated intra-patient dose escalations of 10 and 30 µg / kg during the first week, followed by escalations of 100 µg / kg (10 / 30 / 100). Using this regimen, the predicted C123-NKCE levels after a single dose were [not specified in the original text]. eoi It is about one log lower than the target concentration (Figure 7B) while retaining the efficacy of a fixed dose of 100 µg / kg (Figure 7A).

[0555] Based on the mechanistic PK / PD model, the estimated single-dose exposure (Cmax, AUC0-3d) in humans at a NOAEL of 1 / 10 (safety factor) (i.e., 3000 µg / kg / administration after 5 weeks of twice-weekly administration in stone crab macaques) corresponds to human doses ranging from 1000 to 3000 µg / kg. Considering favorable nonclinical safety results, a starting dose of 10 µg / kg would provide a reasonable safety margin, and the estimated human single-dose exposure is 10 µg / kg.

[0556] Example 3. In vivo efficacy of CD123 NKCE in a mouse model.

[0557] To evaluate the in vivo efficacy of CD123 NKCE, a mouse alternative (muNKp46-huCD123) molecule was generated, allowing the NKp46 arm of CD123 NKCE to bind to mouse NKp46. To generate a mouse reactive alternative, the human reactive NKp46 arm of CD123 NKCE was replaced with a form that binds to the mouse NKp46 protein. The human CD123 reactive arm and the human IgG1 Fc domain were retained. The active Fc domain of human IgG1 retained its binding to all mouse FcγRs and was able to recruit mouse NK cells to induce antibody-dependent cytotoxicity (ADCC). The in vivo efficacy of the mouse alternative was evaluated in severely combined immunodeficient (SCID) mice implanted with disseminated human MOLM-13 tumor cells.

[0558] The activity of muNKp46-huCD123 was compared with that of a mimic monoclonal antibody (mAb) (an Fc-engineered anti-CD123 antibody for enhancing ADCC, capable of binding to mouse FcγR and recruiting mouse effector cells). Tumor cells were intravenously inoculated into mice on day 0. Treatment was administered intraperitoneally on day 1 post-tumor implantation. Data were summarized using Kaplan-Maill curves, and differences in survival between groups were evaluated using a Cox model, as presented in Figure 11.

[0559] In the human MOLM-13 dissemination model, muNKp46-huCD123 NKCE induced statistically significant activity at doses of 5, 0.5, 0.25, and 0.05 mg / kg, with a 100% life extension (ILS) percentage and 60% long-term survivors compared to control at doses of 5, 0.5, and 0.25 mg / kg, and a 30% ILS and 10% long-term survivors at a dose of 0.05 mg / kg. In the human MOLM-13 dissemination model, the analog mAb induced statistically significant activity at a dose of 5 mg / kg, with a 70% ILS and 40% long-term survivors. No activity was observed at doses of 0.5, 0.25, and 0.05 mg / kg.

[0560] muNKp46-huCD123 NKCE showed significantly higher activity than its analogue mAb at doses of 0.5 and 0.25 mg / kg, demonstrating the benefits of co-conjugation to NK cells with NKp46 / CD16.

[0561] Further experiments in the same mouse model demonstrated that antitumor activity was NK cell-dependent. Controls were also evaluated, including an isotype control antibody (NKp46-X) ​​binding to muNKp46 and mouse FcγR but not huCD123, and a second isotype control antibody (X-huCD123) binding to huCD123 and mouse FcγR but not mouse NKp46. As shown in Figure 12, muNKp46-huCD123, which binds to NK cells via mFcγR and NKp46, exhibited higher activity compared to the X-huCD123 isotype control that induces NK cells via mFcγR alone. Antibody-mediated depletion of mouse NK cells led to a significant loss of antitumor activity in both NKCEs, confirming that NK cells participate as effector cells in muNKp46-huCD123.

[0562] Example 4. Pharmacokinetics and pharmacodynamics of CD123 NKCE in stone crab macaques after a single dose.

[0563] Pharmacokinetics

[0564] The aim of this study was to determine the CD123 NKCE PK parameters in monkeys from plasma concentrations following a single administration (0.5, 3, and 3000 µg / kg intravenously (1-hour infusion) or 3.6 and 1000 µg / kg subcutaneously). Toxicity (TK) parameters were calculated after repeated weekly administrations at 100 and 3000 µg / kg for 4 weeks, twice weekly administrations for 5 weeks, and twice weekly subcutaneous administrations at 3000 µg / kg. The CD123 NKCE PK parameters obtained from plasma concentrations after a single 1-hour intravenous infusion and subcutaneous administration are presented in Table 5 below. Plasma concentrations at the end of the infusion (C0.5 µg / kg to 3000 µg / kg) following a single 1-hour intravenous infusion (from 0.5 µg / kg to 3000 µg / kg) are also presented. eoi The increase in exposure (AUC) exceeded expectations on a dose-proportion basis, which is because TMDD exhibits saturation at the highest dose.

[0565] Table 5. Pharmacokinetic parameters in stone crab macaques (mean, CV).

[0566]

[0567] Abbreviation: C max : The maximum concentration observed; t max Time to the observed maximum concentration; AUC: Area under the curve; CL: Total plasma clearance; Vss: Steady-state volume of distribution;

[0568] • n = 1, because the presence of ADA (anti-drug antibody) hinders the accurate estimation of PK parameters.

[0569] Following a single intravenous administration (1-hour infusion) to stone crab macaques, target-mediated drug disposal (TMDD) was demonstrated after infusions of CD123 NKCE from the lowest dose (0.5 µg / kg) to 3 µg / kg. At higher doses (3000 µg / kg intravenously and 1000 µg / kg subcutaneously), target saturation was observed, allowing an estimated linear clearance of CD123 NKCE of 6 mL / day / kg, with a distribution volume of approximately 50 mL / kg and a terminal half-life of 6 days. (Note that PK parameters are sometimes extrapolated, as the ADA response may lead to an underestimation of t1 / 2z; using a PK / PD model constructed in NHP, t1 / 2z was evaluated as 8 days.)

[0570] Following a single subcutaneous administration (1000 µg / kg), the maximum plasma concentration of CD123NKCE was observed between 24 and 48 hours post-administration, and the estimated bioavailability at the target saturation dose (based on AUC0–7d) was close to 35%.

[0571] Following single intravenous infusion (3000 µg / kg) and subcutaneous administration (1000 µg / kg), the effect of anti-drug antibodies (ADA) on exposure was observed at high doses, characterized by a sharp decrease in concentrations 7–10 days after both intravenous and subcutaneous administration. At low doses, TMDD resulted in concentrations below the limit of quantitation on day 7.

[0572] Pharmacodynamics

[0573] For total cells and basophils, a dose-response effect on CD123+ depletion was demonstrated after CD123 NKCE infusion (from 0.5 to 3000 μg / kg), appearing rapidly from the time of first sampling (1.5 hours after the start of infusion), lasting up to 5 hours (i.e., 0.21 days) at 0.5 µg / kg, and up to 14 days and 28 days at 3 and 3000 µg / kg, respectively.

[0574] Plasma concentration (Cg) at the end of the infusion following a single 1-hour intravenous infusion (from 0.5 µg / kg to 3000 µg / kg). eoi The increase in exposure (AUC) exceeded the expected dose-proportionality, which is because TMDD exhibits saturation at the highest dose. See Table 6.

[0575] Table 6. Summary of pharmacokinetic parameters in monkeys. Continuous sampling: n = 2, intravenous; n = 3, subcutaneous; plasma.

[0576]

[0577] Abbreviations: ADA: Anti-drug antibody; AUC: Area under concentration in plasma; C eoi : Concentration observed at the end of infusion; CL: Total plasma clearance; C max : Maximum observed concentration; CV: Coefficient of variation (in parentheses); F%: Using AUC 0-7d Absolute bioavailability (expressed as a percentage) calculated as follows: (34,400 days ng / mL after subcutaneous administration of 1000 µg / kg and 310,000 days ng / mL after intravenous administration of 3000 µg / kg); NC: Not calculated; NA: Not applicable; Vss: Steady-state volume of distribution after a single dose; t max Time of the observed maximum concentration

[0578] Following a single subcutaneous administration (3.6 and 1000 µg / kg), peak plasma concentrations of CD123 NKCE were observed between 6 and 48 hours post-administration. The estimated bioavailability (F%) of CD123 NKCE at 1000 µg / kg was approximately 35% depending on the dose. After 5 weeks of subcutaneous administration at 3000 µg / kg twice weekly, the mean bioavailability ranged from approximately 40% to 65% on days 1, 4, and 8. It can be noted that, using a PK / PD model, the F was 48%, and the absorption rate (kJ / g) was... a The value is 0.078 h. -1 .

[0579] Example 5. In vitro cytokine release assay in whole blood-water phase.

[0580] The potential of CD123 NKCE to induce cytokine release (CR) was evaluated in a whole blood-aqueous phase (WB-AQ) cytokine release assay (CRA), an in vitro human assay designed to inform the potential risk of developing CRS in humans.

[0581] The study included a negative control (untreated whole blood) and a positive control (humanized anti-CD52 IgG, tested at 100 µg / mL) that is known to mediate CR by binding to CD16 on NK cells and is most sensitive to whole blood CRA. The study also included a related tool compound with a similar mechanism of action (depletion of CD123-positive cells) (a bispecific T-cell binder (TCE) antibody (CD3 / CD123)) as a positive control (tested at 10 µg / mL).

[0582] The study was conducted using whole blood from a cohort of 13 healthy donors. CD123 NKCEs were evaluated using a wide concentration range from 0.000025 to 100 µg / mL. Cytokine concentrations (pg / mL) of IFNγ, TNFα, IL-2, IL-13, IL-6, GM-CSF, IL-4, IL-8, MIP1β, and IL-10 were measured in plasma from each donor after overnight treatment (approximately 24 hours) followed by centrifugation using the Meso Scale Discovery (MSD) platform. For each donor, the mean (with standard deviation [SD] and median [Q1, Q3]) fold change of each cytokine relative to untreated whole blood (negative control) was calculated. Furthermore, the total number of CD123+ cells in each sample was determined by flow cytometry and expressed as a percentage of the amount present in untreated whole blood, and the mean (SD) was calculated for each treated sample.

[0583] A wide range of variability was observed in the fold increase in cytokine levels across all donors. This is not surprising for whole blood CRAs, which contain all blood cell types and plasma (including innate immunoglobulins and complement proteins), and these factors can influence cytokine levels.

[0584] The mean fold increase in IFNγ, TNFα, and IL-6 observed in 13 donors after treatment with humanized anti-CD52 IgG1 was equal to or close to the reference values ​​provided by NIBSC for whole blood treated with this compound in 35 donors.

[0585] Following treatment with CD123 NKCE, there was no significant increase in GM-CSF, IL-10, IL-13, IL-4, IL-8, and MIP1β. However, increases in IFNγ and IL-6 were observed in human whole blood in vitro, consistent with NK cell conjugation. TNFα (which also increases after NK cell conjugation and treatment with humanized anti-CD52 IgG1) did not show a significant increase following CD123 NKCE treatment. At all evaluated concentrations of CD123 NKCE, observed IFNγ release was lower than that observed with humanized anti-CD52 IgG1 (mean fold change 42), and at concentrations above 0.25 µg / mL (below which the least increase in IL-6 was observed), IL-6 levels were similar to those induced by humanized anti-CD52 IgG1 (mean fold change 12). Furthermore, when the tested CD123 NKCE concentrations were above 0.25 µg / mL (up to 100 µg / mL), the increases in both IL-6 and IFNγ tended to plateau and did not increase further.

[0586] For comparison, and because significant donor-to-donor variability was observed in most of the cytokines evaluated, median fold change was also calculated. Using median fold change, IL-6 increased only at the two highest CD123 NKCE concentrations tested (50 and 100 µg / mL).

[0587] The cytokine release observed in the CD123 NKCE case was significantly lower than that observed in the CD3-CD123 TCE tool compound case, in which all evaluated cytokines were increased (mean fold change range of 2 to >5000 for 13 donors).

[0588] At CD123 NKCE concentrations above 0.0025 µg / mL, a reduction in CD123+ cell numbers was observed, indicating the expected pharmacology of CD123 NKCE. As expected, despite a significant increase in multiple cytokines, no reduction in CD123+ cell numbers was observed in the humanized anti-CD52IgG1 context. In contrast, the CD3-CD123 TCE tool compound exhibited a high fold increase in all measured cytokines and a reduction in CD123+ cells of approximately 20% at the single concentration tested (10 µg / mL), demonstrating that under the conditions of this study, NK cell synergistic binding with CD123 (rather than T cells) resulted in much less cytokine release and a more effective reduction in CD123+ cells (approximately 50%).

[0589] CD123 NKCE was evaluated in whole blood from 13 healthy donors in vitro using a WB-AQ CRA. At concentrations above 0.25 µg / mL, it resulted in a mean increase in IL-6 (13- to 21-fold) and IFNγ (10- to 24-fold) and induced a reduction in CD123+ cells, consistent with NK cell conjugation and the expected pharmacology of CD123 NKCE. Based on these results, CD123 NKCE demonstrates a low risk of inducing CRS in humans.

[0590] Example 6. An exploratory repeated-dose (once a week for 4 weeks) 1-hour intravenous infusion toxicity study was conducted in NHP, with an observation period of 3 weeks.

[0591] The aim of this study was to determine the potential toxicity of CD123 NKCEs administered weekly via 1-hour intravenous infusion to rhesus macaques for 4 weeks, and to assess the potential delayed onset toxicity and / or reversibility of potential toxicities up to 4 weeks after the last administration. Furthermore, systemic exposure to CD123 NKCEs, the incidence of ADA, and PD parameters (i.e., CD123+ cell depletion and effects on NK cells) were assessed in peripheral blood and bone marrow using flow cytometry.

[0592] Stone crab macaques (35 to 49 months old; 2 animals of each sex and per group) received CD123 NKCE aqueous solution at a dose of 100 or 3000 µg / kg / administered via intravenous infusion over 1 hour, once weekly for 4 weeks (on days 1, 8, 15, and 22; administration volume: 5 mL / kg). Parameters evaluated included mortality, clinical signs, body weight, injection site examination, body temperature, electrocardiographic parameters, hematology, clinical chemistry (including C-reactive protein and ferritin), coagulation, and urinalysis.

[0593] Blood was collected for TK evaluation, ADA, cytokine analysis (IL-2, IL-10, TNFα, IFNγ, IL-1β, IL-6, and IL-8) and flow cytometry analysis.

[0594] Bone marrow samples were also collected for analysis using flow cytometry to evaluate immune function.

[0595] At the end of treatment (i.e., 1 week after the last administration), one monkey of each sex and each dose was euthanized and necropsy was performed, and the remaining monkeys were euthanized and necropsy was performed at the end of the 4-week recovery period. Organ weights were recorded and microscopic examinations were performed.

[0596] The individual TK parameters of CD123 NKCE in plasma after 4 weeks of intravenous infusion at 100 µg / kg / administration for 1 hour each week are presented in Table 7.

[0597] Table 7. TK parameters of CD123NKCE in plasma of individuals (n = 2 / sex) after 4 weeks of intravenous infusion at 100 µg / kg / administration for 1 hour per week.

[0598]

[0599] Abbreviations: LLOQ: Below the limit of quantitation (LLOQ = 0.250 ng / mL); NC: Not calculable

[0600] Individual TK parameters of CD123 NKCE in plasma after 4 weeks of intravenous infusion at 3000 µg / kg / administration for 1 hour per week are presented in Table 8.

[0601] Table 8. TK parameters of CD123NKCE in plasma of individuals (n = 2 / sex) after 4 weeks of intravenous infusion at 3000 µg / kg / administration for 1 hour per week.

[0602]

[0603] Regardless of dose level, the maximum CD123 NKCE plasma concentration was observed between the end of the 1-hour intravenous infusion and 5 hours after the start of each weekly infusion.

[0604] Regardless of the dose level, all monkeys treated with CD123 NKCE showed an ADA-positive status on day 29, except for one animal (male 5) administered 3000 µg / kg, in which no ADA response was observed.

[0605] Overall, CD123 NKCE exposure was significantly reduced in all monkeys after the third infusion due to the presence of ADA, except for animals administered 3000 µg / kg / day (male 5), which showed sustained exposure for 4 weeks, consistent with the negative ADA status throughout the study.

[0606] Following the first infusion on day 1, CD123 NKCE exposure (AUC0–7d) exceeded the dose ratio at doses ranging from 100 to 3000 µg / kg / administration. A 30-fold increase in dose (100–3000 µg / kg / administration) resulted in an increase in exposure greater than 200-fold in both male and female monkeys.

[0607] Overall, after 4 weeks of continuous intravenous infusion of CD123 NKCE at 100 or 3000 µg / kg / week for 1 hour, no accumulation was observed in animals (including those not showing an ADA response) on days 8, 15, and 22. No sex effect was demonstrated on day 1 regardless of dose level (other dosing days were not evaluable due to the animals' positive ADA status).

[0608] Throughout the study, CD123 NKCE was well clinically tolerated, with no changes in clinical signs, weight, or body temperature, or effects on ECG. No adverse changes related to CD123 NKCE were observed in clinical chemistry parameters, and the only such adverse changes were minimal to slight decreases in urea levels in all animals on day 2 (returning to normal by day 29 or 50). No compound-related results were reported in hematological, coagulation, or urinalysis parameters. CD123 NKCE was well tolerated at the injection site.

[0609] In all treated animals (including those administered 3000 µg / kg / day, which were ADA-negative throughout the study and had the highest exposure to CD123 NKCE), very low release of IL-2, IL-6, and IL-10 cytokines was observed after both doses. Changes in IL-6 (< 200 pg / mL) were observed after each administration from 100 µg / kg / day, and changes in IL-2 (< 10 pg / mL) and IL-10 (< 60 pg / mL) were observed primarily after the 3rd and 4th administrations at 3000 µg / kg / day. TNFα, IFNγ, IL-1β, and IL-8 were found below the lower limit of quantitation (LLOQ) in all animals.

[0610] Immunophenotyping using flow cytometry showed similar effects on CD123-positive and NKp46-positive NK cell populations in blood and bone marrow at both tested doses. It should be noted that, under the study conditions, i) the results for CD123-positive monocytes and CD107a-expressing NK cells were not interpreted due to the absence or low cell counts in whole blood and bone marrow; and ii) the number of NK cells expressing PD1 and CD69 was very low and therefore could not be considered as related activation.

[0611] The effects on immune cells were consistent with the expected PD effect of CD123 NKCE, characterized primarily by depletion of CD123-positive cells and a decrease in NKp46-positive NK cells. Changes in the blood included i) a decrease in CD123-positive cells observed in all monkeys from 1.5 hours after the first administration to 24 hours after the third administration (except for males administered 100 μg / kg / day), with a rebound (above baseline) observed in all monkeys on days 22 / 29 (except for males of the highest dose who had no exposure to ADA until the end of dosing, where a rebound was observed at the next time point in the analysis 4 weeks after the last (fourth) administration); and ii) a decrease in NKp46-positive NK cells observed in all monkeys from 1.5 hours to 72 hours after the first administration and 24 hours after the second administration. This decrease was also observed in all monkeys at 1.5 hours after the third administration and in males of the fifth administration at 24 hours after the last (fourth) administration. Changes in the bone marrow included i) a decrease in CD123-positive cells observed 24 hours after the second administration in all monkeys, with a rebound (above baseline) observed in all monkeys (except monkeys 1 and 2 administered 100 μg / kg / day) at the next time point of analysis (one week after the last (fourth) administration); and ii) a decrease in NKp46-positive NK cells observed 24 hours after the second administration in all monkeys, returning to baseline by day 29 (for most monkeys) and day 50 (for male monkey 5).

[0612] Examination of hematoxylin-eosin stained tissues and / or organs revealed microscopic findings limited to bone marrow collected from the sternum and stomach. Microscopic findings in the bone marrow included a minimal to slight increase in the bone marrow cell / erythrocyte (M / E) ratio, characterized by a minimal increase in bone marrow cell count and a decrease in adipocyte count, without atypia. No difference in CD123 immunostaining in sternal bone marrow was observed between treated NHP and reserve control NHP. Given that bone marrow is the classic source of hematopoietic stem cells, and that this study was conducted in healthy animals, we hypothesize that the reduction in CD123+ cells induced by CD123 NKCEs generates a feedback mechanism leading to bone marrow stimulation. Gastric lesions without atypia or ulceration were observed in a small number of animals at both doses, characterized by microfocal glandular monocellular necrosis in the gastric fundus / pyloric mucosa at 1 week after the last (4th) injection in one animal at each dose, and at 4 weeks after the last (4th) injection in both males and females administered 3000 μg / kg / day, along with mild to moderate diffuse mononuclear inflammatory cell infiltration (consisting of a mixed cell population of CD3, CD8, CD68, CD56, and CD123 cells) as revealed by IHC staining in the mucosa. Such results are typically observed in control rhesus monkeys with some degree of gastric inflammation / inflammatory cell infiltration as a background. Evaluation with specific staining agents (Warthin-Starry, Gram, PAS, and toluidine blue) revealed no increase in the opportunistic bacterial and / or fungal populations compared to those typically observed in control NHP.

[0613] The small group size and lack of parallel controls in the study made it difficult to interpret the findings clearly. Nevertheless, gastritis associated with microglandular hyperplasia was considered non-adverse due to its extremely low severity and the absence of relevant clinicopathological changes. A possible relationship with CD123 NKCE was assessed in a GLP toxicity study, which included a large number of animals, including controls.

[0614] Example 7. A study of the toxicity of repeated doses of GLP (twice a week for 5 weeks) administered intravenously or subcutaneously over 1 hour in stone crab macaques, with a recovery period of 6 weeks.

[0615] The aim of this study was to determine the potential toxicity of CD123 NKCE when administered twice weekly for 5 weeks (a total of 9 administrations on days 1, 4, 8, 11, 15, 18, 22, 25, and 29) to rhesus monkeys via intravenous (1-hour infusion) or subcutaneous (bolus) administration. Delayed onset toxicity and / or reversibility of toxicity were assessed during the 1-week observation period and the 6-week recovery period. The TK characteristics, ADA, and PD (CD123+ cell depletion and its effect on NK cells in peripheral blood and bone marrow) profile of CD123 NKCE were also evaluated.

[0616] On days 1, 4, 8, 11, 15, 18, 22, 25, and 29, five rhesus macaques of each sex and each dose received a control solution (10 mM histidine buffer pH 6.0 / sucrose 8% [w / v] / PS80 [w / v] / EDTA 10 µM) subcutaneously (bolus, 2.7 mL / kg) followed by intravenous administration (1 hour infusion, 5 mL / kg) or a solution of CD123 NKCE at doses of 100 and 3000 µg / kg administered intravenously (5 mL / kg) or at doses of 3000 µg / kg administered subcutaneously (2.7 mL / kg).

[0617] One week after the last administration (day 36), three monkeys of each sex and each dose were euthanized and post-mortem examined. At the end of the 6-week recovery period (days 71 / 72), the remaining two monkeys of each sex and each dose were euthanized and post-mortem examined.

[0618] This study evaluated the following parameters and endpoints: clinical signs, local tolerance, body weight, food consumption, body temperature, neurobehavioral assessment, ECG, blood pressure, respiration, ophthalmology, clinicopathological parameters (hematology, coagulation, clinical chemistry, and urinalysis), TK parameters, ADA assessment, cytokine assays, whole blood and bone marrow immunophenotyping, gross findings, organ weight, and histopathological findings.

[0619] The time-to-kine (TK) of plasma CD123 NKCE was evaluated on days 1, 4, 8, and 29, and the summary of plasma CD123 NKCE TK parameters is presented in Table 9.

[0620] Table 9. Mean (%CV, n = 5 / sex / group) CD123 NKCE toxicokinetics in rhesus macaque plasma on days 1, 4, 8, and 29 after 5 weeks of continuous intravenous infusion (100 and 3000 µg / kg / administration) or subcutaneous administration (3000 µg / kg / administration) of CD123 NKCE twice weekly for 1 hour.

[0621]

[0622] Regardless of dose level, route of administration, or sex, most animals treated with CD123 NKCE were ADA-positive by day 15 (26 out of 30 animals), and all animals treated with CD123 NKCE showed ADA-positive status by day 36. Overall, the data indicate that the presence of ADA significantly affected plasma concentrations in all animals, with CD123 NKCE plasma concentrations generally decreasing or becoming unquantifiable by day 15. By day 29, almost all CD123 NKCE plasma concentrations were below the limit of quantification (< 5.00 ng / mL). Between day 29 and day 1, only one male (100 µg / kg / IV) with comparable plasma CD123 NKCE levels was ADA-positive. Due to the presence of ADA affecting exposure, dose-proportioning, sex effect, and bioavailability could not be evaluated on day 29.

[0623] Following intravenous infusion, CD123 NKCE exposure (mean AUC(0–3d) and AUC(0–4d)) in both males and females increased in a greater-than-dose proportion as the dose increased from 100 µg / kg / administration to 3000 µg / kg / administration. Within a 30-fold dose range, mean AUC(0–3d) or AUC(0–4d) values ​​in both males and females increased by 73.4-fold to 86.0-fold on days 1, 4, and / or 8.

[0624] Following repeated intravenous infusions or subcutaneous administration of CD123 NKCE, the mean AUC (0–3d) on days 4 or 8 was generally slightly higher (approximately 1.7 to 2.7 times higher) compared to day 1. On day 29, most TK parameters were unreportable due to a significant decrease in CD123 NKCE plasma concentrations (potentially related to the presence of ADA), and when reportable, the maximum concentration (Cmax) and AUC (0–3d) on day 29 were generally significantly lower compared to day 1. The only exception was one male administered 100 µg / kg intravenously, which was ADA-positive, and the exposure was comparable between day 1 and day 29.

[0625] No sex effect was observed between male and female exposures on days 1, 4, and 8, at different dose levels or routes of administration.

[0626] Following subcutaneous administration of CD123 NKCE at 3000 µg / kg, the mean bioavailability of CD123 NKCE in males and females (assessed by mean AUC(0–3d)) ranged from 41.2% to 63.8% on days 1, 4, and 8.

[0627] No deaths occurred during the study. CD123 NKCE was well tolerated locally regardless of the route of administration and dose level. No clinical signs related to CD123 NKCE were observed in any group, and it had no effect on body weight, food consumption, or clinicopathological parameters, and no results were observed in ophthalmological evaluation.

[0628] No acute or long-term CD123 NKCE-related effects were observed in any group in terms of neurobehavioral response, body temperature, arterial blood pressure, quantitative and qualitative ECG parameters, or respiratory rate evaluated on days 4 and 25. Significant effects on IL-1Ra, IL-10, TNFα, and MIP1β levels were observed only at 3000 µg / kg / administration when administered intravenously, and these significant effects included minimal to moderate increases in IL-1Ra, IL-10, and MIP1β at 1 or 5 hours after the 5th and 9th infusions, and a minimal increase in TNFα at 1 hour after the 9th infusion. Maximum concentrations (IL-10, TNFα, and MIP1β) were reached at 1 hour (IL-1Ra) and 5 hours (IL-1Ra) after the 9th administration. These changes were considered non-adverse, taking into account the magnitude of the changes and the return of levels to baseline at 5 or 24 hours.

[0629] Changes in the total CD123+ cell population and NK cells expressing NKp46 were observed in blood and bone marrow, and said changes included (i) a CD123 NKCE-related decrease in the total CD123+ cell count in all treatment groups from the first administration to 24 hours after the third administration in blood and in bone marrow 24 hours after the third administration, followed by a CD123 NKCE-related increase in both blood and bone marrow, and a return to baseline in both blood and bone marrow at the end of the 6-week recovery period; and (ii) a CD123 NKCE-related decrease in the number of NKp46+ NK cells in blood from the first administration to 24 hours after the first administration and after the third administration; this decrease was followed by a CD123 NKCE-related increase in the intravenous dose group, and a return to baseline at the end of the 6-week recovery period; in bone marrow, no CD123 NKCE-related effect on NKp46+ NK cells was observed.

[0630] No CD123 NKCE-related organ weights, gross or microscopic findings were observed at terminal euthanasia (day 36, one week after the last 9th administration) and at the end of the 6-week recovery period. Mild-to-severe inflammatory cell infiltration in the gastric mucosa was observed in a few animals, including control males; these changes (known to occur commonly in untreated stone crab macaques (20, 21, 22)) were considered to be within the range of background changes and unrelated to compound administration.

[0631] NOAEL was set at 3000 µg / kg / administration via intravenous and subcutaneous routes. The mean AUC (0–3d) values ​​(after the 3rd administration) for males and females after intravenous / subcutaneous administration were 261,000 / 167,000 days.ng / mL and 257,000 / 140,000 days.ng / mL, respectively.

[0632] Example 8. Study of CD123 NKCE in patients with relapsed or refractory (r / r) AML, B-ALL, BPDCN, or high-risk myelodyplastic syndromes.

[0633] This is a phase 1 / 2, open-label, first-in-human study to determine the maximum tolerated dose or maximum administered dose and to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity of CD123 NKCE as described herein in a variety of hematologic malignancies in adult or pediatric patients.

[0634] Research Overview:

[0635] This is a first-in-human, open-label phase 1 / 2 dose-escalation / extension phase 1 / 2 study to determine the maximum tolerated dose or maximum administered dose and to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity of intravenously administered CD123 NKCE as a single agent in participants aged ≥12 years with relapsed or refractory AML (r / r AML), B-ALL, or HR-MDS, or in pediatric patients with r / r AML, BPDCN, or B-ALL.

[0636] principle

[0637] Mounting evidence suggests that AML originates from leukemia stem cells (LSCs), which generate leukemia blasts in in vitro and in vivo models. It is hypothesized that the persistence of LSCs leads to relapse after initial remission. Therefore, eradication of LSCs may be a necessary condition for cure and is an important therapeutic target. One potential therapeutic target for AML is the surface receptor CD123, the interleukin (IL)-3 receptor α chain (IL-3Rα) highly expressed on LSCs. CD123 is overexpressed in AML blasts and CD34-positive / CD38-negative AML leukemia stem cells, while other hematologic malignancies, including MDS and B-ALL, are also CD123-positive. In normal peripheral blood, CD123 exhibits varying levels of expression, with the highest density on plasmacytoid dendritic cells and basophils, and moderate levels on myeloid dendritic cells, monocytes, eosinophils, and immature B cells.

[0638] The proposed study is a first-in-human, dose-finding study to determine the safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of intravenous (IV) administration of CD123 NKCE to adults and adolescents with CD123-expressing hematologic malignancies who have relapsed or failed prior treatment and have no alternative standard treatment options.

[0639] This is a first-in-human (FIH) phase 1 dose-escalation study designed to determine the optimal dose and regimen (Recommended Dose for Extension, RDE) of CD123-NKCE that can be safely administered in patients with AML. The proposed starting dose of 10 µg / kg is based on a mechanism-based pharmacokinetic (PK) / pharmacodynamic (PD) model and takes into account the estimated exposure in humans (Cmax, AUC0-3d), equivalent to 1 / 10 of the exposure quantified at the administration-defined No Observed Affect Level (NOAEL) of 3000 µg / kg after 5 weeks of twice-weekly administration in non-human primates (NHP) (safety factor).

[0640] Study duration

[0641] If a participant does not meet the criteria for discontinuing treatment with the investigational drug (IMP), the expected duration of this Phase 1 / 2 study for that participant is approximately 2.5 years. The study duration includes:

[0642] • A screening period of up to 2 weeks (14 days) in incremental / expansion (except for 28 days for MDS cohort B).

[0643] • 3 months of induction therapy (3 cycles, 28 days per cycle), 2 years of maintenance therapy (13 cycles, 56 days per cycle), and

[0644] • End of Treatment (EOT) follow-up after the last administration of the investigational drug (IMP) (30 days ± 7 days). If discontinuation occurs during the maintenance period, the EOT is preferably performed within 30 days (+ / - 7 days), but must be performed within 60 days (+ / - 7 days) of the last IMP administration or before starting further therapy, whichever comes first.

[0645] Survival status will be collected for each participant during follow-up periods of 2 months ± 14 days to assess survival. The study will be completed when the last participant in the extended portion is followed for 12 months or until death (whichever comes first) to record extended remission and 1-year survival.

[0646] Even if CR, CRi, or CRh is achieved before completing 3 cycles (28 days each), trial participants will still receive 3 induction cycles. Based on an evaluation of available safety data, including DLT, AE, SAE, clinical PK, and PD, the study committee may recommend increasing or decreasing the number of induction cycles. If CR, CRi, or CRh (AML participants) or CR, CR equivalence, or PR (MDS participants) is not achieved within 3 induction cycles, induction therapy must continue until CR, CRi, or CRh (AML participants) or CR, CR equivalence, or PR (MDS participants) is achieved (up to a maximum of 6 cycles). Once a trial participant achieves CR, CRi, or CRh, the participant must continue maintenance cycles (56 days each) up to a total of 13 cycles. Participants who achieve a adequate response to CD123 NKCE treatment and meet other criteria for hematopoietic stem cell transplantation (HSCT) may continue HSCT after discontinuation of CD123 NKCE. If a participant discontinues CD123 NKCE for HSCT but is subsequently deemed ineligible for HSCT, the participant may restart CD123 NKCE treatment at the originally planned DL and dose.

[0647] Stop criteria

[0648] If a trial participant experiences an unacceptable adverse event or evidence of disease progression, or if the trial participant decides to discontinue CD123 NKCE treatment, the trial participant must discontinue CD123 NKCE treatment.

[0649] Stop rules:

[0650] “Excessive toxicity” was defined as death (excluding PD-related deaths) or a Grade 4 treatment-induced adverse event (TEAE) occurring within 30 days of treatment. Bayesian toxicity monitoring was used to determine the stoppage boundary based on the following assumptions: maximum permissible excessive toxicity probability = 0.2, prior distribution of excessive toxicity rate: β(1,1), minimum number of participants before applying the early stop rule = 6, cohort size = 1, and a probability of at least 80% after an excessive toxicity rate > 0.2. Table 21 shows the number of participants with excessive toxicity observed before enrollment in the trial, at which point the trial was suspended until the research committee appropriately evaluated the causes of death and toxicity and established a correction plan.

[0651] Table 9B - Bayesian toxicity monitoring program during dose extension period.

[0652]

[0653] Abbreviation: N = total number of participants evaluated; n = number of participants with excessive toxicity.

[0654] Research Intervention

[0655] Investigational drug (AxMP):

[0656] • Formulation: The CD123 NKCE drug product is presented as a powder in an infusion solution in vials containing 100 mg / vial.

[0657] • One or more routes of administration: intravenous infusion. Specific instructions on the preparation and administration of CD123 NKCE are provided in the pharmacy manual.

[0658] • Dosing regimen:

[0659] Induction: Depending on the dose level, CD123 NKCE will be administered twice and / or once weekly during cycle 1. In subsequent induction cycles (2+), CD123 NKCE will be administered once weekly, i.e., on days 1, 8, 15, and 22 of a 28-day cycle. Participants will receive at least 3 induction cycles before switching to maintenance therapy.

[0660] Adult escalation: Administer CD123 NKCE only on days 4 and 11 of cycle 1, and for lower dose levels requiring twice-weekly administration during the first two weeks, administer on days 1, 4, 8, 11, 15, and 22 of cycle 1. Administer CD123 NKCE once weekly on days 1, 8, 15, and 22 of all other cycles.

[0661] Adult extension: CD123 NKCE is administered on D1, D8, D15 and D22 of all cycles.

[0662] Pediatric escalation / expansion: Administer CD123 NKCE on days 1, 8, 15, and 22 of all cycles.

[0663] Maintenance (Adult and Pediatric Escalation / Extended): Once participants achieve CR, CRh, or CRi, CD123 NKCE will be administered intravenously on days 1 and 29 of a 56-day cycle, continuing for up to 13 cycles. The frequency of IMP treatment may be adjusted during the maintenance period based on emerging clinical data. As additional clinical data becomes available, the maintenance dosing schedule within the maintenance cycle may be further adjusted to potentially include more frequent or less frequent dosing within the cycle.

[0664] • The intravenous administration regimen will be based on weight; however, if a participant weighs > 133 kg, the upper limit of the participant's weight for dosage calculation will be 133 kg.

[0665] Non-experimental drugs:

[0666] For the first cycle, prior to CD123 NKCE administration, participants will receive the following prophylactic medication to prevent or reduce the severity of potential acute adverse events:

[0667] • Acetaminophen (paracetamol) 650 to 1000 mg orally (preferably orally, but intravenous administration is acceptable) or adult / pediatric equivalent, then administered as needed thereafter.

[0668] • Administer CD123 NKCE (preferably intravenously, but oral administration is also acceptable when intravenous preparations are unavailable) or pediatric equivalents (e.g., diphenhydramine, chlorpheniramine, dextromethorphan, depending on local approval and availability) 30–60 minutes before administration (intravenous administration of diphenhydramine 25 to 50 mg, followed by administration as needed thereafter).

[0669] Starting from cycle 2, prophylactic administration may be as needed, as described below:

[0670] • For participants who did not experience CRS or IRR during the first cycle: Pre-administration is optional for subsequent infusions at the investigator's discretion. However, if a participant experiences any level of CRS / IRR during subsequent infusions without pre-administration, pre-administration must be restarted with all subsequent infusions.

[0671] • If a participant experiences a CRS / IRR of < 3 only during the first cycle and then does not experience any other CRS / IRR during their second cycle, the investigator may consider omitting pre-administration for the next cycle. If no CRS / IRR is observed in the next cycle without pre-administration, pre-administration is optional for subsequent cycles at the investigator's discretion. However, if a participant experiences any level of CRS / IRR in the next cycle without pre-administration, pre-administration must be restarted in all subsequent cycles.

[0672] Follow-up

[0673] All ongoing or newly reported related adverse events, as well as all SAEs (regardless of causation), should be followed up at least every 30 days until they subside or stabilize.

[0674] The first cutoff date will be at the end of the first cycle of the last participant in the treated and evaluable dose-escalation portion of DLT, so as to evaluate data from at least the first cycle of all participants when determining MTD, MAD, and preliminary RDE.

[0675] The second and subsequent cutoff dates will be when the last participant in the extended / optimized cohort A, cohort B, or cohort D completes three induction cycles, experiences early progression, or achieves CRc (for AML) or CR, CR equivalence, PR, CRL, CRh, or HI (for HR-MDS) (whichever occurs first) to assess malignancy response. Different data extraction dates may be applied based on adult and pediatric cohort recruitment times and data maturity. After the second cutoff date, if clinical benefit is observed, participants may continue IMP until permanent discontinuation criteria are met (personal choice, adverse effects, no response, pregnancy, or low body weight), and all assessments will continue according to the study activity schedule. The study completion date will be when the last participant in the extended cohort is followed for 12 months or until death (whichever occurs first) to document prolonged response and 1-year survival.

[0676] Statistical considerations:

[0677] • Analysis of primary endpoints

[0678] - Dose escalation and dose expansion / optimization cohort C: DLTs will be summarized by dose level and overall. Detailed information on DLTs will be provided by the participants.

[0679] - Dose expansion / optimization cohorts A, B, and D: For cohorts A and D, descriptive statistics will be used to summarize the CRc rate (for R / R AML). For cohort B, descriptive statistics will be used to summarize the overall response rate. The 90% two-sided confidence interval will be calculated using the Clopper-Pearson method.

[0680] • Analysis of secondary endpoints

[0681] - Dose escalation and dose expansion / optimization cohort C: Descriptive statistics using the Clopper-Pearson method with 90% two-sided confidence intervals will be used to summarize the CR + CRh + CRi rates for R / R AML and B-ALL, the CR rate for HR-MDS in dose escalation, the ORR rate for HR-MDS in dose expansion / optimization cohort C, and the CR + clinical CR rate for BPDCN (if applicable).

[0682] - Dose Escalation and Expansion / Optimization: Descriptive statistics will be used to summarize adverse events / severity events (AEs) and laboratory abnormalities, as well as PK profile characteristics, that occurred during the duration of treatment, by dose level (for escalation), cohort (for expansion), and overall population (if applicable). PK profile characteristics and immunogenicity will be assessed by dose level / cohort and overall population.

[0683] - Dosage Extension / Optimization Section, Cohorts A, B, and D: For cohorts A and D, descriptive statistics will be used to summarize the CR + CRh rate, CR + CRh + CRi + MLFS + PR rate, and subsequent HSCT rate for AML. A 90% two-sided confidence interval will be calculated using the Clopper-Pearson method. The time to the event endpoint, such as the duration of different response types (CR + CRh + CRi; CR + CRh; and CR + CRi + CRh + MLFS + PR), EFS, TTF, and OS, will be summarized using the Kaplan-Mail method. For cohort B, descriptive statistics will be used to summarize the alternative CR rate and subsequent HSCT rate. A 90% two-sided confidence interval will be calculated using the Clopper-Pearson method. The time to the event endpoint, such as ORR duration, alternative CR duration, OS, EFS, TTF, and PFS, will be summarized using the Kaplan-Mail method.

[0684] Goals and End Points

[0685] Table 10.

[0686] Target end Major dose escalation: Adults: Determine the maximum tolerated dose (MTD) or maximum administered dose (MAD) and one or more recommended doses (RDE) for escalation of CD123NKCE as a single agent in participants with relapsed or refractory acute myeloid leukemia (r / r AML), high-risk myelodyplastic syndrome (HR-MDS), or B-cell acute lymphoblastic leukemia (B-ALL). Pediatrics: Determine the maximum tolerated dose (MTD) or maximum administered dose (MAD) and RDE of CD123NKCE as a single agent in participants with r / r AML, blastic plasmacytoid dendritic cell tumor (BPDCN), or B-ALL. Incidence of dose-limiting toxicities (DLTs) during Cycle 1 (Day 1 to Day 28) Expanded Cohort C: To confirm the tolerability of CD123 NKCE in Japanese participants. Expanded / Optimized: (Cohorts A, B, and D) To evaluate the anticancer activity of CD123 NKCE as a single agent administered at one or more of the initial recommended doses (RDE) for expansion, and to confirm the RDE in participants with r / r AML and MDS. AML: Composite Complete Remission (CRc) Rate: The proportion of participants with CR (complete remission) + CRh (CR with partial hematologic recovery) + CRi (complete remission with incomplete hematologic recovery) according to the modified AML IWG 2003 response criteria. MDS: Overall Response Rate (CR + CR Equivalent + PR + CRL + CRh + HI) - IWG2023 MDS Response Criteria Secondary (adult and pediatric) tasks include determining the initial RDE (incremental portion) and confirming the RDE (expanded / optimized portion; cohorts A, B, and D). Overall safety and tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary anticancer activity. Characterizing the overall safety and tolerability profile of CD123 NKCE (incremental and extended / optimized) The type, frequency, severity, and relationship of adverse events or abnormalities to CD123 NKCE observed in laboratory tests or investigations; discontinuation of medication due to adverse events. Characterizing the PK spectrum of CD123 NKCE as a single agent (incremental and expanded / optimized) <![CDATA[PK parameters of CD123 NKCE (at least C 谷 ).]]> Evaluation of the potential immunogenicity of CD123 NKCE (incremental and expanded / optimized) Incidence of anti-drug antibodies (ADA) Preliminary evidence for assessing hematological response (excluding BPDCN) (incremental and expanded / optimized cohort C) • AML: CR + CRh + CRi rate according to the International Working Group (IWG) response standard as modified by AML 2003. • MDS: CR rate of the incremental portion according to the IWG 2006 MDS response standard and ORR rate of the extended / optimized queue C according to the IWG 2023 MDS response standard. • B-ALL: CR + CRh + CRi rate as defined by NCCN. Additional parameters for evaluating the antitumor activity of CD123 NKCE under RDE in AML participants (extended / optimized portion; cohorts A and D) According to the modified AML IWG response criteria, the overall response rate is defined as the proportion of participants with CR or Cri or CRh or PR or MLFS (morphologically leukemia-free status). The duration of CR + CRh + Cri (duration of CRc) is defined as the time interval from the first recorded evidence of CRc (CR, CRh, or CRi) until disease relapse or death from any cause (whichever occurred first). The duration of CR + Cri + CRh + PR + MLFS (duration of overall response rate) is defined as the time from the first recorded evidence of CR or Cri or CRh or PR or MLFS until disease relapse or death from any cause (whichever occurred first). The alternative CR rate is defined as the proportion of participants with CR + CRh (complete remission with partial hematologic recovery). The duration of CR + CRh (duration of alternative CR) is defined as the time from the first recorded evidence of CR or CRh until disease relapse or death from any cause (whichever occurred first). Event-free survival (EFS) is defined as the time interval from day one of treatment allocation to the date of the earliest evidence of relapse, treatment failure, or death. Overall survival is defined as the time interval from day one of treatment allocation to death from any cause. The rate of hematopoietic stem cell transplantation (HSCT) performed immediately after administration of study treatment but before subsequent therapy for AML. Time to treatment failure (TTF) is defined as the time from day one of treatment allocation to discontinuation due to any cause other than remission, such as disease relapse, refractory disease, participant preference, or death. Evaluation of transfusion-independent rate (TI) after CD123 NKCE treatment under RDE (expanded / optimized, cohort A and cohort D) The conversion rate of transfusion dependence during the 56-day period after baseline was the proportion of participants who were transfusion-free at baseline and remained transfusion-free during the 56-day period after baseline. Additional parameters for evaluating the anticancer activity of CD123 NKCE under RDE in MDS participants (expansion / optimization, cohort B). <![CDATA[The alternative CR rate is defined as the proportion of participants with CR, CR equivalent, CRuni, CRbi, and CRh. Duration of ORR: As per the 2023 IWG recommendations, it is defined as the time interval from the first documented evidence of CR, CR equivalent, CR L , CRh, PR, or HI to PD or to recurrence of CR, CR equivalent, CR L , CRh, PR, or HI or death from any cause (whichever occurs first). Event-free survival (EFS): It is measured from the first day of treatment assignment to the occurrence of the first of the following events: PD, i.e., failure to achieve CR (or CR equivalent), PR, CR L , CRh, or HI within 6 months of entering the study. Recurrence of CR (or CR equivalent), PR, CR L , CRh, or HI. Death from any cause. Patients known not to have experienced any of the stated events are censored at the last date known not to have experienced any of the stated events. Overall survival is defined as the time interval from the first day of treatment assignment to death from any cause. Rate of hematopoietic stem cell transplantation (HSCT) procedures performed immediately after administration of study treatment but before subsequent therapy. Time to treatment failure (TTF) is defined as the time interval from the first day of treatment assignment to discontinuation due to any cause other than remission (e.g., disease recurrence, disease progression, unacceptable AE, participant preference, or death). Duration of alternative CR (CR + CR equivalent + CRL + CRh): As per the 2023 IWG recommendations, the time interval from the first documented evidence of CR, CR equivalent, CRL, or CRh until PD or to recurrence of CR, CR equivalent, CRL, or CRh or death from any cause (whichever occurs first). Progression-free survival (PFS): It is defined as the time interval from the first day of treatment assignment to PD, to recurrence of CR (or CR equivalent), PR, CRL, CRh, or HI, to death from any cause (whichever occurs first).]]> Exploratory Pediatrics only: Preliminary evidence for assessing hematological response (incremental) For BPDCN participants, anti-leukemic activity (e.g., ORR, clinical CR rate) is defined by the International Harmonization Project (IHP) Lymphoma Criteria (modified). Monitoring CD123 expression and its dynamics under treatment CD123 expression in various leukemia blast and non-blast populations in blood and bone marrow will be assessed using multiparametric flow cytometry and time-series analysis of baseline and treatment-related changes in CD123 receptor density. Assess minimal residual disease (MRD) in participants who achieve CR, CRh, or Cri, and correlate MRD with clinical outcomes (AML extension / optimization only, cohorts A, C, and D). Minimal residual disease was assessed in the bone marrow and / or blood of participants who achieved CR, CRh, or Cri using molecular biological evaluation and / or flow cytometry, and correlated with clinical outcomes. Assess NK and T cell subsets (e.g., CD4, CD8) and their activation status. Monitor NK and T cell subsets (e.g., CD4, CD8) and their activation status in peripheral blood and bone marrow aspirates. This study investigates the relationship between parameters such as CD123 expression, disease molecular subtypes (e.g., defined by biomarker expression, cytogenetics and / or genomics / total transcriptology), baseline IgG, different AML subgroups (i.e., PIF / ER and LR), and clinical response. Assess the potential correlation between baseline disease-specific features (CD123 expression, molecular subtype, PIF / ER vs. LR, cytogenetics and / or genomics / total transcriptomics), baseline IgG, and clinical outcomes. The percentage decrease in CD123+ blasts was measured during dose escalation and expansion. The kinetics of the decrease in the percentage of treatment-induced CD123+ blasts (as a pharmacodynamic marker of response to IMP) were assessed. Assess the levels of cytokines after treatment and their relationship with safety profile and clinical outcomes. Blood plasma cytokine levels were assessed at specific time points after treatment administration to evaluate the potential association between cytokine levels and safety and clinical outcomes. Assess participants' health-related quality of life (HRQL), disease and treatment-related symptoms, and the impact of symptoms (expansion / optimization period, cohort A and cohort C). The participant-reported outcome tool was used to assess quality of life in acute myeloid leukemia (AML-QOL). The Patient Global Severity Impression (PGIS) and Patient Global Change Impression (PGIC) scales were used as anchors to estimate / confirm clinically significant changes in scores identified in the AML-QOL / domain scores. Qualitative assessment of patients' treatment history (expansion / optimization period, cohorts A, B, and C) The results from the Treatment Qualitative Assessment Version 2 (PQAT-v2) were used to assess participants’ perceived treatment advantages and disadvantages.

[0687] Participants

[0688] Escalation: CD123 will be administered to participants with r / r AML, HR-MDS, B-ALL, or BPDCN. The dose-escalation portion is expected to recruit approximately 66 adult participants with evaluable DLTs and assess approximately 11 major DLs, with intermediate DLs potentially investigated if dose or schedule changes are made. The pediatric dose-escalation portion is expected to recruit approximately 24 participants and assess approximately 3 major DLs, with intermediate DLs potentially investigated if dose or schedule changes are made. Actual sample size will vary depending on the observed DLTs, the number of DLs actually explored, and the number of participants recruited with non-evaluable DLTs.

[0689] Table 11. Number of participants during the incremental period.

[0690]

[0691] The preliminary RDE (pRDE) determined during the dose escalation phase can be further evaluated during the expansion / optimization phase in the first 10-20 participants recruited in the pRDE.

[0692] Expanding / Optimizing: Approximately 100 r / r AML patients are expected to be recruited and treated in cohort A; up to 70 participants with HR-MDS are expected to be treated in cohort B; up to 9 participants with HR-MDS, B-ALL, or AML are expected to be treated in cohort C; and up to 80 pediatric participants with r / r AML are expected to be treated in cohort D. In the informal interim analysis, Bayesian methods will be used for ongoing safety and efficacy monitoring. The sample size in the dose expansion portion is not based on efficacy calculations but is intended to provide preliminary information on the activity of CD123 NKCE as a single agent in adult r / r AML, HR-MDS, and pediatric r / r AML populations.

[0693] Inclusion criteria: Participants are eligible for inclusion in the study only if all of the following criteria apply:

[0694] A. Age

[0695] Participants must be ≥ 1 year old (except ≥ 2 years old in France) when the informed consent form is signed by the trial participant or legal guardian, and will be allocated as follows:

[0696] a. Adult group: Age ≥ 12 years old.

[0697] b. Pediatric Group: Age 1 year (except ≥ 2 years in France) to 17 years. Participants aged 12-17 years may be recruited as the second or subsequent participant in DL ≥ 3, or into the “Pediatric Group”, as determined by the research committee based on supporting data obtained in accordance with the protocol.

[0698] Table 12.

[0699]

[0700] Participants aged 12-17 years may be recruited as the second or subsequent participant in the DL to enter DL ≥ 3, or into the “Pediatric Group”, as determined by the research committee based on the supporting data obtained in accordance with the protocol.

[0701] B. Increasing and expanding cohorts A, C, and D (adults and pediatrics)

[0702] a. Diagnosed with primary or secondary AML [any subtype] according to the World Health Organization (WHO) classification. Participants with AML must meet one of the following criteria i), ii), or iii), and are limited to those who are not receiving a treatment with known clinical benefit (or are ineligible).

[0703] i. Initial induction failure (PIF) AML, defined as one of the following refractory diseases:

[0704] 1. Intensive induction attempts at each institution. Induction attempts include high-dose and / or standard-dose cytarabine ± anthracyclines / anthracyclines ± antimetabolites, with or without growth factors or containing targeted therapy. Examples include, but are not limited to: a one-cycle regimen containing high-dose cytarabine (HiDAC); a one-cycle regimen of liposomal cytarabine and donomycin; or a two-cycle regimen containing standard-dose cytarabine.

[0705] 2. For adults 75 years of age or older, or adults ...

Claims

1. A method for treating or preventing leukemia or myelodysplastic syndrome in a subject in need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46, wherein the binding protein is administered to the subject at a dose of at least 3 µg / kg.

2. A method for treating or preventing leukemia or myelodysplastic syndrome in a subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46, wherein the first antigen-binding domain comprises: The heavy chain variable domain (VH1) comprises CDR-H1, H2, and H3 corresponding to amino acid sequences of SEQ ID NO: 1, 2, and 3, or corresponding to amino acid sequences of SEQ ID NO: 4, 5, and 5, respectively; and The light chain variable domain (VL1) contains CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 7, 8 and 9, respectively, or corresponding to the amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively. The binding protein was administered to the subject at a dose of at least 3 µg / kg.

3. The method according to claim 1 or 2, wherein the dose is between about 3 µg / kg and about 6000 µg / kg.

4. The method according to any one of claims 1-3, wherein the dose is between about 3 µg / kg and about 30 µg / kg.

5. The method according to any one of claims 1-3, wherein the dose is between about 10 µg / kg and 100 µg / kg.

6. The method according to any one of claims 1-3, wherein the dose is between about 30 µg / kg and 100 µg / kg.

7. The method according to any one of claims 1-3, wherein the dose is between about 13 µg / kg and about 130 µg / kg.

8. The method according to any one of claims 1-3, wherein the dose is between about 15 µg / kg and about 150 µg / kg.

9. The method according to any one of claims 1-3, wherein the dose is between about 20 µg / kg and about 200 µg / kg.

10. The method according to any one of claims 1-3, wherein the dose is between about 30 µg / kg and about 300 µg / kg.

11. The method according to any one of claims 1-3, wherein the dose is between about 40 µg / kg and about 400 µg / kg.

12. The method according to any one of claims 1-3, wherein the dose is between about 45 µg / kg and about 450 µg / kg.

13. The method according to any one of claims 1-3, wherein the dose is between about 60 µg / kg and about 600 µg / kg.

14. The method according to any one of claims 1-3, wherein the dose is between about 100 µg / kg and about 1000 µg / kg.

15. The method according to any one of claims 1-3, wherein the dose is between about 300 µg / kg and about 1000 µg / kg.

16. The method according to any one of claims 1-3, wherein the dose is between 400 µg / kg and about 1300 µg / kg.

17. The method according to any one of claims 1-3, wherein the dose is between about 450 µg / kg and about 1500 µg / kg.

18. The method according to any one of claims 1-3, wherein the dose is between about 600 µg / kg and about 1000 µg / kg.

19. The method according to any one of claims 1-3, wherein the dose is between about 600 µg / kg and about 1500 µg / kg.

20. The method according to any one of claims 1-3, wherein the dose is between about 600 µg / kg and about 2000 µg / kg.

21. The method according to any one of claims 1-3, wherein the dose is between about 1000 µg / kg and about 3000 µg / kg.

22. The method according to any one of claims 1-3, wherein the dose is between about 1300 µg / kg and about 3000 µg / kg.

23. The method according to any one of claims 1-3, wherein the dose is between about 1500 µg / kg and about 3000 µg / kg.

24. The method according to any one of claims 1-3, wherein the dose is between about 2000 µg / kg and about 3000 µg / kg.

25. The method according to any one of claims 1-3, wherein the dosage is about 3 µg / kg.

26. The method according to any one of claims 1-3, wherein the dosage is about 10 µg / kg.

27. The method according to any one of claims 1-3, wherein the dosage is about 13 µg / kg.

28. The method according to any one of claims 1-3, wherein the dosage is about 15 µg / kg.

29. The method according to any one of claims 1-3, wherein the dosage is about 20 µg / kg.

30. The method according to any one of claims 1-3, wherein the dosage is about 30 µg / kg.

31. The method according to any one of claims 1-3, wherein the dosage is about 40 µg / kg.

32. The method according to any one of claims 1-3, wherein the dosage is about 45 µg / kg.

33. The method according to any one of claims 1-3, wherein the dosage is about 50 µg / kg.

34. The method according to any one of claims 1-3, wherein the dosage is about 60 µg / kg.

35. The method according to any one of claims 1-3, wherein the dosage is about 100 µg / kg.

36. The method according to any one of claims 1-3, wherein the dosage is about 130 µg / kg.

37. The method according to any one of claims 1-3, wherein the dosage is about 150 µg / kg.

38. The method according to any one of claims 1-3, wherein the dosage is about 200 µg / kg.

39. The method according to any one of claims 1-3, wherein the dosage is about 300 µg / kg.

40. The method according to any one of claims 1-3, wherein the dosage is about 400 µg / kg.

41. The method according to any one of claims 1-3, wherein the dosage is about 450 µg / kg.

42. The method according to any one of claims 1-3, wherein the dosage is about 600 µg / kg.

43. The method according to any one of claims 1-3, wherein the dosage is about 750 ug / kg.

44. The method according to any one of claims 1-3, wherein the dosage is about 1000 µg / kg.

45. The method according to any one of claims 1-3, wherein the dosage is about 1300 µg / kg.

46. ​​The method according to any one of claims 1-3, wherein the dosage is about 1500 µg / kg.

47. The method according to any one of claims 1-3, wherein the dosage is about 2000 µg / kg.

48. The method according to any one of claims 1-3, wherein the dosage is about 3000 µg / kg.

49. The method according to any one of claims 1-3, wherein the dosage is about 4000 µg / kg.

50. The method according to any one of claims 1-3, wherein the dose is about 4500 µg / kg.

51. The method according to any one of claims 1-3, wherein the dosage is about 6000 µg / kg.

52. The method according to any one of the preceding claims, wherein the binding protein is administered intravenously, subcutaneously, intraperitoneally, or intramuscularly to the subject.

53. The method of claim 52, wherein the binding protein is administered intravenously to the subject.

54. The method according to any one of the preceding claims, wherein the binding protein is applied to the subject for at least one cycle.

55. The method of claim 54, wherein the at least one cycle is about 28 days.

56. The method of claim 52 or 53, wherein the binding protein is administered to the subject for at least two cycles.

57. The method according to any one of claims 52-56, wherein the binding protein is administered to the subject for three or more cycles.

58. The method of claim 1, wherein the first antigen-binding domain having binding specificity to CD123 comprises: The heavy chain variable domain (VH1) comprises CDR-H1, H2, and H3 corresponding to amino acid sequences of SEQ ID NO: 1, 2, and 3, or corresponding to amino acid sequences of SEQ ID NO: 4, 5, and 5, respectively; and The light chain variable domain (VL1) contains CDR-L1, L2 and L3 corresponding to amino acid sequences of SEQ ID NO: 7, 8 and 9, or amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively.

59. The method according to any one of the preceding claims, wherein: a. The VH1 contains at least about 90% of the amino acid sequence identical to the amino acid sequence of SEQ ID NO: 41, and the VL1 contains at least about 90% of the amino acid sequence identical to the amino acid sequence of SEQ ID NO: 43; or b. The VH1 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 42, and the VL1 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

44.

60. The method according to any one of the preceding claims, wherein: a. The VH1 comprises the amino acid sequence of SEQ ID NO: 41, and the VL1 comprises the amino acid sequence of SEQ ID NO: 43; or b. The VH1 contains the amino acid sequence of SEQ ID NO: 42, and the VL1 contains the amino acid sequence of SEQ ID NO:

44.

61. The method according to any one of the preceding claims, wherein the second antigen-binding domain having binding specificity to NKp46 comprises: a. The second heavy chain variable domain (VH2) contains CDRs H1, H2, and H3 corresponding to the following amino acid sequences: i. Specifically, SEQ ID NO: 13, 14 and 15; ii. Specifically, SEQ ID NO: 16, 17, and 18; iii. Specifically, SEQ ID NO: 19, 20, and 21; iv. respectively, SEQ ID NO: 22, 23 and 24; or v. respectively, SEQ ID NO: 16, 25 and 26; and b. The second light chain variable domain (VL2) contains CDRs-L1, L2, and L3 corresponding to the following amino acid sequences: i. Specifically, SEQ ID NO: 27, 28, and 29; ii. Specifically, SEQ ID NO: 30, 31 and 32; iii. Specifically, SEQ ID NO: 33, 34 and 35; iv. respectively, SEQ ID NO: 36, 37 and 38; or v. respectively, SEQ ID NO: 39, 31 and 40.

62. The method according to any one of the preceding claims, wherein: a. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 45, and the VL1 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 53; b. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 46, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 54; c. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 47, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 55; d. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 48, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 56; e. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 49, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 57; f. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 50, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 58; g. The VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 51, and wherein the VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 59; or h. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 52, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

60.

63. The method according to any one of the preceding claims, wherein: a. The VH2 comprises the amino acid sequence of SEQ ID NO: 45, and the VL1 comprises the amino acid sequence of SEQ ID NO: 53; b. The VH2 comprises the amino acid sequence of SEQ ID NO: 46, and the VL2 comprises the amino acid sequence of SEQ ID NO: 54; c. The VH2 comprises the amino acid sequence of SEQ ID NO: 47, and the VL2 comprises the amino acid sequence of SEQ ID NO: 55; d. The VH2 comprises the amino acid sequence of SEQ ID NO: 48, and the VL2 comprises the amino acid sequence of SEQ ID NO: 56; e. The VH2 comprises the amino acid sequence of SEQ ID NO: 49, and the VL2 comprises the amino acid sequence of SEQ ID NO: 57; f. The VH2 comprises the amino acid sequence of SEQ ID NO: 50, and the VL2 comprises the amino acid sequence of SEQ ID NO: 58; g. The VH2 comprises the amino acid sequence of SEQ ID NO: 51, and the VL2 comprises the amino acid sequence of SEQ ID NO: 59; or h. The VH2 comprises the amino acid sequence of SEQ ID NO: 52, and the VL2 comprises the amino acid sequence of SEQ ID NO:

60.

64. The method according to any one of the preceding claims, wherein the binding protein comprises three polypeptide chains (I), (II), and (III) forming two ABDs, as defined below: V 1A - C 1A - Hinge 1 - (C H 2-C H 3) A (I) V 1B - C 1B - Hinge 2 - (C H 2-C H 3) B - L1 - V 2A - C 2A - Hinge 3(II) V 2B - C 2B (III) in: V 1A and V 1B Forming binding pairs V1 (V H1 / V L1 ); V 2A and V 2B Forming binding pairs V2 (V H2 / V L2 ); C 1A and C 1B Forming a pair of C1 (C H 1 / C L ), and C 2A and C 2B Formation of C2 (C H 1 / C L ), where C H 1 represents the immunoglobulin heavy chain constant domain 1, and C L It is the constant structural domain of the immunoglobulin light chain; Hinge 1, hinge 2 and hinge 3 may be the same or different, and correspond to all or part of the immunoglobulin hinge region; (C H 2-C H 3) A and (C) H 2-C H 3) B For the same or different, and containing the immunoglobulin heavy chain constant domain 2 (C H 2) and immunoglobulin heavy chain constant domain 3 (C H 3); L1 is an amino acid linker.

65. The method according to claim 64, wherein: C 1B For immunoglobulin heavy chain constant domain 1 (C H 1); C 2A For immunoglobulin heavy chain constant domain 1 (C H 1); C L Corresponding to the immunoglobulin κ light chain constant domain (C κ ); (C H 2-C H 3) A The amino acid sequence corresponding to SEQ ID NO: 69; (C H 2-C H 3) B The amino acid sequence corresponding to SEQ ID NO: 70; Hinge 1 corresponds to the amino acid sequence of SEQ ID NO: 74; Hinge 2 corresponds to the amino acid sequence of SEQ ID NO: 75; Hinge 3 corresponds to the amino acid sequence of SEQ ID NO: 77; L1 corresponds to the amino acid sequence of SEQ ID NO:

76.

66. The method according to any one of the preceding claims, wherein residue N297 of the Fc region or a variant thereof, according to the EU number, comprises N-linked glycosylation.

67. The method according to any one of the preceding claims, wherein all or part of the Fc region or a variant thereof binds to the human CD16A (FcγRIII) polypeptide.

68. The method according to any one of claims 64-67, wherein the method comprises at least two polypeptide chains linked by at least one disulfide bond.

69. The method according to claim 68, wherein the polypeptide chain (I) and the polypeptide chain (II) are separated by C 1A At least one disulfide bond is connected between hinge 2 and the polypeptide chain (II) and the polypeptide chain (III) via hinge 3 to C. 2B At least one disulfide bond connects them.

70. The method according to any one of claims 64-69, wherein V 1A For V L1 And V 1B For V H1 .

71. The method according to any one of claims 64-70, wherein V 2A For V H2 And V 2B For V L2 .

72. The method according to any one of the preceding claims, wherein: (a) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 13; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 14; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 15; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 27; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 28; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

29. (b) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 17; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 18; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO:30; the CDR-L2 contains the amino acid sequence of SEQ ID NO:31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

32. (c) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 19; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 20; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 21; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO:33; the CDR-L2 contains the amino acid sequence of SEQ ID NO:34; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

35. (d) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 22; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 23; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 24; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO:36; the CDR-L2 contains the amino acid sequence of SEQ ID NO:37; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

38. (e) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 25; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 26; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO:39; the CDR-L2 contains the amino acid sequence of SEQ ID NO:31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

40. (f) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 10; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 11; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 13; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 14; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 15; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 27; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 28; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

29. (g) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 10; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 11; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 17; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 18; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 30; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

32. (h) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 10; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 11; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 19; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 20; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 21; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 33; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 34; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

35. (i) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 10; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 11; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 22; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 23; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 24; V L2 The CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; the CDR-L2 containing the amino acid sequence of SEQ ID NO: 37; the CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; or (j) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 10; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 11; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 25; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 26; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 39; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

40.

73. The method according to any one of the preceding claims, wherein: (a) V H1 and V L1 The amino acid sequences corresponding to SEQ ID NO: 41 and 43, or the amino acid sequences corresponding to SEQ ID NO: 42 and 44, respectively; and / or (b) V H2 and V L2 Corresponding to The amino acid sequences of SEQ ID NO: 45 and 53, respectively; The amino acid sequences of SEQ ID NO: 46 and 54, respectively; The amino acid sequences of SEQ ID NO: 47 and 55, respectively; The amino acid sequences of SEQ ID NO: 48 and 56, respectively; The amino acid sequences of SEQ ID NO: 49 and 57, respectively; The amino acid sequences of SEQ ID NO: 50 and 58, respectively; Specifically, the amino acid sequences of SEQ ID NO: 51 and 59; or The amino acid sequences of SEQ ID NO: 52 and 60, respectively.

74. The method according to claim 73, wherein: (q)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 45; V L2 Contains the amino acid sequence of SEQ ID NO: 53; (r)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 46; V L2 The amino acid sequence containing SEQ ID NO: 54; (s)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 47; V L2 Contains the amino acid sequence of SEQ ID NO: 55; (t)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 48; V L2 Contains the amino acid sequence of SEQ ID NO: 56; (u)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 49; VL2 contains the amino acid sequence of SEQ ID NO:

57. (v)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 50; V L2 The amino acid sequence containing SEQ ID NO: 58; (w)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 51; V L2 Contains the amino acid sequence of SEQ ID NO: 59; (x)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 52; VL2 contains the amino acid sequence of SEQ ID NO:

60. (y)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 45; VL2 contains the amino acid sequence of SEQ ID NO:

53. (z)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 46; V L2 The amino acid sequence containing SEQ ID NO: 54; (aa)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 47; V L2 Contains the amino acid sequence of SEQ ID NO: 55; (bb)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 48; V L2 Contains the amino acid sequence of SEQ ID NO: 56; (cc)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 49; V L2 The amino acid sequence containing SEQ ID NO: 57; (dd)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 50; V L2 The amino acid sequence containing SEQ ID NO: 58; (ee)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 51; V L2 Contains the amino acid sequence of SEQ ID NO: 59; or (ff)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 52; V L2 It contains the amino acid sequence of SEQ ID NO:

60.

75. The method according to any one of claims 64-74, wherein: -The polypeptide (I) consists of the amino acid sequence of SEQ ID NO: 64; -The polypeptide (II) consists of the amino acid sequence of SEQ ID NO: 65; and -The polypeptide (III) consists of the amino acid sequence of SEQ ID NO:

66.

76. The method according to any one of the preceding claims, wherein the leukemia is acute myeloid leukemia (AML).

77. The method of claim 76, wherein the AML is relapsed or refractory.

78. The method according to any one of the preceding claims, wherein the leukemia is B-cell acute lymphoblastic leukemia (B-ALL).

79. The method according to any one of the preceding claims, wherein the myelodystrophy syndrome is a high-risk myelodystrophy syndrome (HR-MDS).

80. The method according to any one of the preceding claims, wherein the blood disease or disorder is myelodyplasia syndrome.

81. The method of claim 80, wherein the myelodystrophy is high-risk myelodystrophy (HR-MDS).

82. A method for treating or preventing leukemia or myelodysplastic syndrome in a subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46. The first antigen-binding domain comprises a heavy chain variable domain (VH1) and a light chain variable domain (VL1), wherein: The VH1 contains complementarity-determining regions (CDRs) - H1, H2, and H3 corresponding to the amino acid sequences of SEQ ID NO: 1, 2, and 3; and The VL1 comprises CDR-L1, L2, and L3 corresponding to the amino acid sequences of SEQ ID NO: 7, 8, and 9; and The second antigen-binding domain comprises a heavy chain variable domain (VH2) and a light chain variable domain (VL2), wherein: The VH2 contains CDR-H1, H2, and H3 corresponding to the amino acid sequences of SEQ ID NO: 13, 14, and 15; and The VL2 comprises CDR-L1, L2, and L3 corresponding to the amino acid sequences of SEQ ID NO: 27, 28, and 29; Furthermore, all or part of the Fc region of the immunoglobulin or its variants bind to the human Fc-γ receptor. The binding protein was administered to the subject at a dose of at least 3 µg / kg.

83. A method for treating or preventing leukemia or myelodysplastic disorder in a subject in need, the method comprising administering a binding protein to the subject, the binding protein comprising all or part of a first antigen-binding domain having binding specificity to CD123, a second antigen-binding domain having binding specificity to NKp46, and an immunoglobulin Fc region or a variant thereof that binds to a human Fc-γ receptor, wherein the binding protein comprises: A polypeptide (I) consisting of the amino acid sequence of SEQ ID NO: 64; A polypeptide (II) consisting of the amino acid sequence of SEQ ID NO: 65; and A polypeptide (III) consisting of the amino acid sequence of SEQ ID NO:

66. The binding protein was administered to the subject at a dose of at least 3 µg / kg.

84. A method for treating or preventing leukemia or hematologic malignancy in a pediatric subject in need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46.

85. A method for treating or preventing blastic plasmacytoid dendritic cell tumor (BPDCN) in a subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46.

86. A method for treating or preventing blastic plasmacytoid dendritic cell tumor (BPDCN) in a subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46, wherein the first antigen-binding domain comprises: The heavy chain variable domain (VH1) comprises CDR-H1, H2, and H3 corresponding to amino acid sequences of SEQ ID NO: 1, 2, and 3, or corresponding to amino acid sequences of SEQ ID NO: 4, 5, and 5, respectively; and The light chain variable domain (VL1) contains CDR-L1, L2 and L3 corresponding to amino acid sequences of SEQ ID NO: 7, 8 and 9, or amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively.

87. The method according to any one of claims 84-86, wherein the subject is a pediatric subject.

88. The method according to any one of claims 84-87, wherein the binding protein is administered intravenously, subcutaneously, intraperitoneally, or intramuscularly to the subject.

89. The method of claim 88, wherein the binding protein is administered intravenously to the subject.

90. The method according to any one of claims 84-89, wherein the binding protein is administered at a dose between about 500 µg / kg and about 12,000 µg / kg.

91. The method according to any one of claims 84-90, wherein the binding protein is administered at a dose between about 600 µg / kg and about 1000 µg / kg.

92. The method according to any one of claims 84-91, wherein the binding protein is administered at a dose between about 600 µg / kg and about 1500 µg / kg.

93. The method of claim 90, wherein the dosage is about 500 µg / kg.

94. The method of claim 90, wherein the dosage is about 600 µg / kg.

95. The method of claim 90, wherein the dosage is about 1000 µg / kg.

96. The method of claim 90, wherein the dosage is about 1500 µg / kg.

97. The method of claim 90, wherein the dosage is about 3000 µg / kg.

98. The method of claim 90, wherein the dosage is about 6000 µg / kg.

99. The method of claim 90, wherein the dosage is about 12000 µg / kg.

100. The method according to any one of claims 84-99, wherein the first antigen-binding domain having binding specificity to CD123 comprises: The heavy chain variable domain (VH1) comprises CDR-H1, H2, and H3 corresponding to amino acid sequences of SEQ ID NO: 1, 2, and 3, or corresponding to amino acid sequences of SEQ ID NO: 4, 5, and 5, respectively; and The light chain variable domain (VL1) contains CDR-L1, L2 and L3 corresponding to amino acid sequences of SEQ ID NO: 7, 8 and 9, or amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively.

101. The method according to any one of the preceding claims, wherein: a. The VH1 contains at least about 90% of the amino acid sequence identical to the amino acid sequence of SEQ ID NO: 41, and the VL1 contains at least about 90% of the amino acid sequence identical to the amino acid sequence of SEQ ID NO: 43; or b. The VH1 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 42, and the VL1 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

44.

102. The method according to any one of the preceding claims, wherein: a. The VH1 comprises the amino acid sequence of SEQ ID NO: 41, and the VL1 comprises the amino acid sequence of SEQ ID NO: 43; or b. The VH1 contains the amino acid sequence of SEQ ID NO: 42, and the VL1 contains the amino acid sequence of SEQ ID NO:

44.

103. The method according to any one of the preceding claims, wherein the second antigen-binding domain having binding specificity to NKp46 comprises: a. The second heavy chain variable domain (VH2) contains CDRs H1, H2, and H3 corresponding to the following amino acid sequences: i. Specifically, SEQ ID NO: 13, 14 and 15; ii. Specifically, SEQ ID NO: 16, 17, and 18; iii. Specifically, SEQ ID NO: 19, 20, and 21; iv. respectively, SEQ ID NO: 22, 23 and 24; or v. respectively, SEQ ID NO: 16, 25 and 26; and b. The second light chain variable domain (VL2) contains CDRs-L1, L2, and L3 corresponding to the following amino acid sequences: i. Specifically, SEQ ID NO: 27, 28, and 29; ii. Specifically, SEQ ID NO: 30, 31 and 32; iii. Specifically, SEQ ID NO: 33, 34 and 35; iv. respectively, SEQ ID NO: 36, 37 and 38; or v. respectively, SEQ ID NO: 39, 31 and 40.

104. The method according to any one of the preceding claims, wherein: a. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 45, and the VL1 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 53; b. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 46, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 54; c. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 47, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 55; d. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 48, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 56; e. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 49, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 57; f. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 50, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 58; g. The VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 51, and wherein the VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 59; or h. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 52, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

60.

105. The method according to any one of the preceding claims, wherein: a. The VH2 comprises the amino acid sequence of SEQ ID NO: 45, and the VL1 comprises the amino acid sequence of SEQ ID NO: 53; b. The VH2 comprises the amino acid sequence of SEQ ID NO: 46, and the VL2 comprises the amino acid sequence of SEQ ID NO: 54; c. The VH2 comprises the amino acid sequence of SEQ ID NO: 47, and the VL2 comprises the amino acid sequence of SEQ ID NO: 55; d. The VH2 comprises the amino acid sequence of SEQ ID NO: 48, and the VL2 comprises the amino acid sequence of SEQ ID NO: 56; e. The VH2 comprises the amino acid sequence of SEQ ID NO: 49, and the VL2 comprises the amino acid sequence of SEQ ID NO: 57; f. The VH2 comprises the amino acid sequence of SEQ ID NO: 50, and the VL2 comprises the amino acid sequence of SEQ ID NO: 58; g. The VH2 comprises the amino acid sequence of SEQ ID NO: 51, and the VL2 comprises the amino acid sequence of SEQ ID NO: 59; or h. The VH2 comprises the amino acid sequence of SEQ ID NO: 52, and the VL2 comprises the amino acid sequence of SEQ ID NO:

60.

106. The method according to any one of the preceding claims, wherein the binding protein comprises three polypeptide chains (I), (II) and (III) forming two ABDs, as defined below: V 1A - C 1A - Hinge 1 - (C H 2-C H 3) A (I) V 1B - C 1B - Hinge 2 - (C H 2-C H 3) B - L1 - V 2A - C 2A - Hinge 3(II) V 2B - C 2B (III) in: V 1A and V 1B Forming binding pairs V1 (V H1 / V L1 ); V 2A and V 2B Forming binding pairs V2 (V H2 / V L2 ); C 1A and C 1B Forming a pair of C1 (C H 1 / C L ), and C 2A and C 2B Formation of C2 (C H 1 / C L ), where C H 1 represents the immunoglobulin heavy chain constant domain 1, and C L It is the constant structural domain of the immunoglobulin light chain; Hinge 1, hinge 2 and hinge 3 may be the same or different, and correspond to all or part of the immunoglobulin hinge region; (C H 2-C H 3) A and (C) H 2-C H 3) B For the same or different, and containing the immunoglobulin heavy chain constant domain 2 (C H 2) and immunoglobulin heavy chain constant domain 3 (C H 3); L1 is an amino acid linker.

107. The method according to claim 106, wherein: C 1B For immunoglobulin heavy chain constant domain 1 (C H 1); C 2A For immunoglobulin heavy chain constant domain 1 (C H 1); C L Corresponding to the immunoglobulin κ light chain constant domain (C κ ); (C H 2-C H 3) A The amino acid sequence corresponding to SEQ ID NO: 69; (C H 2-C H 3) B The amino acid sequence corresponding to SEQ ID NO: 70; Hinge 1 corresponds to the amino acid sequence of SEQ ID NO: 74; Hinge 2 corresponds to the amino acid sequence of SEQ ID NO: 75; Hinge 3 corresponds to the amino acid sequence of SEQ ID NO: 77; L1 corresponds to the amino acid sequence of SEQ ID NO:

76.

108. The method according to any one of the preceding claims, wherein residue N297 of the Fc region or a variant thereof, according to the EU number, comprises N-linked glycosylation.

109. The method according to any one of the preceding claims, wherein all or part of the Fc region or a variant thereof binds to the human CD16A (FcγRIII) polypeptide.

110. The method according to any one of claims 106-109, wherein the method comprises at least two polypeptide chains linked by at least one disulfide bond.

111. The method according to claim 110, wherein the polypeptide chain (I) and the polypeptide chain (II) are separated by C 1A At least one disulfide bond is connected between the polypeptide chain (II) and the polypeptide chain (III) via hinge 3 and C. 2B At least one disulfide bond connects them.

112. The method according to any one of claims 106-111, wherein V 1A For V L1 And V 1B For V H1 .

113. The method according to any one of claims 106-111, wherein V 2A For V H2 And V 2B For V L2 .

114. The method according to any one of the preceding claims, wherein: (a) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 13; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 14; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 15; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 27; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 28; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

29. (b) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 17; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 18; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO:30; the CDR-L2 contains the amino acid sequence of SEQ ID NO:31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

32. (c) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 19; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 20; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 21; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO:33; the CDR-L2 contains the amino acid sequence of SEQ ID NO:34; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

35. (d) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 22; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 23; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 24; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO:36; the CDR-L2 contains the amino acid sequence of SEQ ID NO:37; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

38. (e) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 25; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 26; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO:39; the CDR-L2 contains the amino acid sequence of SEQ ID NO:31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

40. (f) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 10; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 11; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 13; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 14; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 15; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 27; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 28; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

29. (g) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 10; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 11; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 17; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 18; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 30; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

32. (h) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 10; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 11; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 19; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 20; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 21; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 33; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 34; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

35. (i) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 10; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 11; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 22; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 23; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 24; V L2 The CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; the CDR-L2 containing the amino acid sequence of SEQ ID NO: 37; the CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; or (j) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 10; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 11; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 25; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 26; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 39; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

40.

115. The method according to any one of the preceding claims, wherein: (a) V H1 and V L1 The amino acid sequences corresponding to SEQ ID NO: 41 and 43, or the amino acid sequences corresponding to SEQ ID NO: 42 and 44, respectively; and / or (b) V H2 and V L2 Corresponding to The amino acid sequences of SEQ ID NO: 45 and 53, respectively; The amino acid sequences of SEQ ID NO: 46 and 54, respectively; The amino acid sequences of SEQ ID NO: 47 and 55, respectively; The amino acid sequences of SEQ ID NO: 48 and 56, respectively; The amino acid sequences of SEQ ID NO: 49 and 57, respectively; The amino acid sequences of SEQ ID NO: 50 and 58, respectively; Specifically, the amino acid sequences of SEQ ID NO: 51 and 59; or The amino acid sequences of SEQ ID NO: 52 and 60, respectively.

116. The method according to claim 115, wherein: (a)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 45; V L2 Contains the amino acid sequence of SEQ ID NO: 53; (b)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 46; V L2 The amino acid sequence containing SEQ ID NO: 54; (c)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 47; V L2 Contains the amino acid sequence of SEQ ID NO: 55; (d)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 48; V L2 Contains the amino acid sequence of SEQ ID NO: 56; (e)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 49; VL2 contains the amino acid sequence of SEQ ID NO:

57. (f)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 50; V L2 The amino acid sequence containing SEQ ID NO: 58; (g)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 51; V L2 Contains the amino acid sequence of SEQ ID NO: 59; (h)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 52; VL2 contains the amino acid sequence of SEQ ID NO:

60. (i)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 45; VL2 contains the amino acid sequence of SEQ ID NO:

53. (j)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 46; V L2 The amino acid sequence containing SEQ ID NO: 54; (k)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 47; V L2 Contains the amino acid sequence of SEQ ID NO: 55; (l)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 48; V L2 Contains the amino acid sequence of SEQ ID NO: 56; (m)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 49; V L2 The amino acid sequence containing SEQ ID NO: 57; (n)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 50; V L2 The amino acid sequence containing SEQ ID NO: 58; (o)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 51; V L2 Contains the amino acid sequence of SEQ ID NO: 59; or (p)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 52; V L2 It contains the amino acid sequence of SEQ ID NO:

60.

117. The method according to any one of claims 106-116, wherein: -The polypeptide (I) consists of the amino acid sequence of SEQ ID NO: 64; -The polypeptide (II) consists of the amino acid sequence of SEQ ID NO: 65; and -The polypeptide (III) consists of the amino acid sequence of SEQ ID NO:

66.

118. A method for treating or preventing leukemia in a pediatric subject in need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46.

119. The method of claim 118, wherein the leukemia is acute myeloid leukemia (AML).

120. The method of claim 119, wherein the AML is relapsed or refractory.

121. The method according to claim 118, wherein the leukemia is B-cell acute lymphoblastic leukemia (B-ALL).

122. The method according to any one of claims 118-121, wherein the binding protein is administered intravenously, subcutaneously, intraperitoneally or intramuscularly to the subject.

123. The method of claim 122, wherein the binding protein is administered intravenously to the subject.

124. The method according to any one of claims 118-123, wherein the first antigen-binding domain having binding specificity to CD123 comprises: The heavy chain variable domain (VH1) comprises CDR-H1, H2, and H3 corresponding to amino acid sequences of SEQ ID NO: 1, 2, and 3, or corresponding to amino acid sequences of SEQ ID NO: 4, 5, and 5, respectively; and The light chain variable domain (VL1) contains CDR-L1, L2 and L3 corresponding to amino acid sequences of SEQ ID NO: 7, 8 and 9, or amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively.

125. The method according to any one of claims 118-124, wherein: a. The VH1 contains at least about 90% of the amino acid sequence identical to the amino acid sequence of SEQ ID NO: 41, and the VL1 contains at least about 90% of the amino acid sequence identical to the amino acid sequence of SEQ ID NO: 43; or b. The VH1 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 42, and the VL1 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

44.

126. The method according to any one of the preceding claims, wherein: a. The VH1 comprises the amino acid sequence of SEQ ID NO: 41, and the VL1 comprises the amino acid sequence of SEQ ID NO: 43; or b. The VH1 contains the amino acid sequence of SEQ ID NO: 42, and the VL1 contains the amino acid sequence of SEQ ID NO:

44.

127. The method according to any one of the preceding claims, wherein the second antigen-binding domain having binding specificity to NKp46 comprises: a. The second heavy chain variable domain (VH2) contains CDRs H1, H2, and H3 corresponding to the following amino acid sequences: i. Specifically, SEQ ID NO: 13, 14 and 15; ii. Specifically, SEQ ID NO: 16, 17, and 18; iii. Specifically, SEQ ID NO: 19, 20, and 21; iv. respectively, SEQ ID NO: 22, 23 and 24; or v. respectively, SEQ ID NO: 16, 25 and 26; and b. The second light chain variable domain (VL2) contains CDRs-L1, L2, and L3 corresponding to the following amino acid sequences: i. Specifically, SEQ ID NO: 27, 28, and 29; ii. Specifically, SEQ ID NO: 30, 31 and 32; iii. Specifically, SEQ ID NO: 33, 34 and 35; iv. respectively, SEQ ID NO: 36, 37 and 38; or v. respectively, SEQ ID NO: 39, 31 and 40.

128. The method according to any one of the preceding claims, wherein: a. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 45, and the VL1 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 53; b. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 46, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 54; c. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 47, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 55; d. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 48, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 56; e. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 49, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 57; f. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 50, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 58; g. The VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 51, and wherein the VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 59; or h. The VH2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 52, and the VL2 contains at least about 90% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

60.

129. The method according to any one of the preceding claims, wherein: a. The VH2 comprises the amino acid sequence of SEQ ID NO: 45, and the VL1 comprises the amino acid sequence of SEQ ID NO: 53; b. The VH2 comprises the amino acid sequence of SEQ ID NO: 46, and the VL2 comprises the amino acid sequence of SEQ ID NO: 54; c. The VH2 comprises the amino acid sequence of SEQ ID NO: 47, and the VL2 comprises the amino acid sequence of SEQ ID NO: 55; d. The VH2 comprises the amino acid sequence of SEQ ID NO: 48, and the VL2 comprises the amino acid sequence of SEQ ID NO: 56; e. The VH2 comprises the amino acid sequence of SEQ ID NO: 49, and the VL2 comprises the amino acid sequence of SEQ ID NO: 57; f. The VH2 comprises the amino acid sequence of SEQ ID NO: 50, and the VL2 comprises the amino acid sequence of SEQ ID NO: 58; g. The VH2 comprises the amino acid sequence of SEQ ID NO: 51, and the VL2 comprises the amino acid sequence of SEQ ID NO: 59; or h. The VH2 comprises the amino acid sequence of SEQ ID NO: 52, and the VL2 comprises the amino acid sequence of SEQ ID NO:

60.

130. The method according to any one of the preceding claims, wherein the binding protein comprises three polypeptide chains (I), (II) and (III) forming two ABDs, as defined below: V 1A - C 1A - Hinge 1 - (C H 2-C H 3) A (I) V 1B - C 1B - Hinge 2 - (C H 2-C H 3) B - L1 - V 2A - C 2A - Hinge 3(II) V 2B - C 2B (III) in: V 1A and V 1B Forming binding pairs V1 (V H1 / V L1 ); V 2A and V 2B Forming binding pairs V2 (V H2 / V L2 ); C 1A and C 1B Forming a pair of C1 (C H 1 / C L ), and C 2A and C 2B Formation of C2 (C H 1 / C L ), where C H 1 represents the immunoglobulin heavy chain constant domain 1, and C L It is the constant structural domain of the immunoglobulin light chain; Hinge 1, hinge 2 and hinge 3 may be the same or different, and correspond to all or part of the immunoglobulin hinge region; (C H 2-C H 3) A and (C) H 2-C H 3) B For the same or different, and containing the immunoglobulin heavy chain constant domain 2 (C H 2) and immunoglobulin heavy chain constant domain 3 (C H 3); L1 is an amino acid linker.

131. The method of claim 130, wherein: C 1B For immunoglobulin heavy chain constant domain 1 (C H 1); C 2A For immunoglobulin heavy chain constant domain 1 (C H 1); C L Corresponding to the immunoglobulin κ light chain constant domain (C κ ); (C H 2-C H 3) A The amino acid sequence corresponding to SEQ ID NO: 69; (C H 2-C H 3) B The amino acid sequence corresponding to SEQ ID NO: 70; Hinge 1 corresponds to the amino acid sequence of SEQ ID NO: 74; Hinge 2 corresponds to the amino acid sequence of SEQ ID NO: 75; Hinge 3 corresponds to the amino acid sequence of SEQ ID NO: 77; L1 corresponds to the amino acid sequence of SEQ ID NO:

76.

132. The method according to any one of the preceding claims, wherein residue N297 of the Fc region or a variant thereof, according to the EU number, comprises N-linked glycosylation.

133. The method according to any one of the preceding claims, wherein all or part of the Fc region or a variant thereof binds to the human CD16A (FcγRIII) polypeptide.

134. The method according to any one of claims 130-133, wherein the method comprises at least two polypeptide chains linked by at least one disulfide bond.

135. The method according to claim 134, wherein the polypeptide chain (I) and the polypeptide chain (II) are separated by C 1A At least one disulfide bond is connected between the polypeptide chain (II) and the polypeptide chain (III) via hinge 3 and C. 2B At least one disulfide bond connects them.

136. The method according to any one of claims 130-135, wherein V 1A For V L1 And V 1B For V H1 .

137. The method according to any one of claims 130-136, wherein V 2A For V H2 And V 2B For V L2 .

138. The method according to any one of the preceding claims, wherein: (a) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 13; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 14; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 15; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 27; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 28; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

29. (b) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 17; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 18; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO:30; the CDR-L2 contains the amino acid sequence of SEQ ID NO:31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

32. (c) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 19; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 20; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 21; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO:33; the CDR-L2 contains the amino acid sequence of SEQ ID NO:34; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

35. (d) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 22; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 23; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 24; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO:36; the CDR-L2 contains the amino acid sequence of SEQ ID NO:37; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

38. (e) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 1; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 2; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 3; V L1 CDR-L1 containing the amino acid sequence of SEQ ID NO: 7; CDR-L2 containing the amino acid sequence of SEQ ID NO: 8; CDR-L3 containing the amino acid sequence of SEQ ID NO: 9; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 25; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 26; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO:39; the CDR-L2 contains the amino acid sequence of SEQ ID NO:31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

40. (f) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 10; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 11; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 13; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 14; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 15; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 27; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 28; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

29. (g) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 10; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 11; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 17; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 18; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 30; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

32. (h) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 10; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 11; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 19; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 20; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 21; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 33; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 34; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

35. (i) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 10; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 11; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 22; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 23; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 24; V L2 The CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; the CDR-L2 containing the amino acid sequence of SEQ ID NO: 37; the CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; or (j) V H1 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 4; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 5; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; V L1 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 10; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 11; the CDR-L3 contains the amino acid sequence of SEQ ID NO: 12; V H2 The CDR-H1 contains the amino acid sequence of SEQ ID NO: 16; the CDR-H2 contains the amino acid sequence of SEQ ID NO: 25; the CDR-H3 contains the amino acid sequence of SEQ ID NO: 26; V L2 The CDR-L1 contains the amino acid sequence of SEQ ID NO: 39; the CDR-L2 contains the amino acid sequence of SEQ ID NO: 31; and the CDR-L3 contains the amino acid sequence of SEQ ID NO:

40.

139. The method according to any one of the preceding claims, wherein: (a) V H1 and V L1 The amino acid sequences corresponding to SEQ ID NO: 41 and 43, or the amino acid sequences corresponding to SEQ ID NO: 42 and 44, respectively; and / or (b) V H2 and V L2 Corresponding to The amino acid sequences of SEQ ID NO: 45 and 53, respectively; The amino acid sequences of SEQ ID NO: 46 and 54, respectively; The amino acid sequences of SEQ ID NO: 47 and 55, respectively; The amino acid sequences of SEQ ID NO: 48 and 56, respectively; The amino acid sequences of SEQ ID NO: 49 and 57, respectively; The amino acid sequences of SEQ ID NO: 50 and 58, respectively; Specifically, the amino acid sequences of SEQ ID NO: 51 and 59; or The amino acid sequences of SEQ ID NO: 52 and 60, respectively.

140. The method of claim 139, wherein: (a)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 45; V L2 Contains the amino acid sequence of SEQ ID NO: 53; (b)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 46; V L2 The amino acid sequence containing SEQ ID NO: 54; (c)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 47; V L2 Contains the amino acid sequence of SEQ ID NO: 55; (d)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 48; V L2 Contains the amino acid sequence of SEQ ID NO: 56; (e)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 49; VL2 contains the amino acid sequence of SEQ ID NO:

57. (f)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 50; V L2 The amino acid sequence containing SEQ ID NO: 58; (g)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 Contains the amino acid sequence of SEQ ID NO: 51; V L2 Contains the amino acid sequence of SEQ ID NO: 59; (h)V H1 Contains the amino acid sequence of SEQ ID NO: 41; V L1 Contains the amino acid sequence of SEQ ID NO: 43; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 52; VL2 contains the amino acid sequence of SEQ ID NO:

60. (i)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 VL2 contains the amino acid sequence of SEQ ID NO: 45; VL2 contains the amino acid sequence of SEQ ID NO:

53. (j)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 46; V L2 The amino acid sequence containing SEQ ID NO: 54; (k)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 47; V L2 Contains the amino acid sequence of SEQ ID NO: 55; (l)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 48; V L2 Contains the amino acid sequence of SEQ ID NO: 56; (m)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 49; V L2 The amino acid sequence containing SEQ ID NO: 57; (n)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 50; V L2 The amino acid sequence containing SEQ ID NO: 58; (o)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 51; V L2 Contains the amino acid sequence of SEQ ID NO: 59; or (p)V H1 Contains the amino acid sequence of SEQ ID NO: 42; V L1 Contains the amino acid sequence of SEQ ID NO: 44; V H2 Contains the amino acid sequence of SEQ ID NO: 52; V L2 It contains the amino acid sequence of SEQ ID NO:

60.

141. The method according to any one of claims 130-140, wherein: -The polypeptide (I) consists of the amino acid sequence of SEQ ID NO: 64; -The polypeptide (II) consists of the amino acid sequence of SEQ ID NO: 65; and -The polypeptide (III) consists of the amino acid sequence of SEQ ID NO:

66.

142. A method for treating or preventing blastic plasmacytoid dendritic cell tumor (BPDCN) in a pediatric subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123 and a second antigen-binding domain having binding specificity to NKp46. The first antigen-binding domain comprises a heavy chain variable domain (VH1) and a light chain variable domain (VL1), wherein: The VH1 contains complementarity-determining regions (CDRs) - H1, H2, and H3 corresponding to the amino acid sequences of SEQ ID NO: 1, 2, and 3; and The VL1 comprises CDR-L1, L2, and L3 corresponding to the amino acid sequences of SEQ ID NO: 7, 8, and 9; and The second antigen-binding domain comprises a heavy chain variable domain (VH2) and a light chain variable domain (VL2), wherein: The VH2 contains CDR-H1, H2, and H3 corresponding to the amino acid sequences of SEQ ID NO: 13, 14, and 15; and The VL2 comprises CDR-L1, L2, and L3 corresponding to the amino acid sequences of SEQ ID NO: 27, 28, and 29; Furthermore, all or part of the Fc region of the immunoglobulin or its variants bind to the human Fc-γ receptor.

143. A method for treating or preventing blastic plasmacytoid dendritic cell tumor (BPDCN) in a pediatric subject of need, the method comprising administering a binding protein to the subject, the binding protein comprising a first antigen-binding domain having binding specificity to CD123, a second antigen-binding domain having binding specificity to NKp46, and all or part of an immunoglobulin Fc region or a variant thereof that binds to a human Fc-γ receptor, wherein the binding protein comprises: A polypeptide (I) consisting of the amino acid sequence of SEQ ID NO: 64; A polypeptide (II) consisting of the amino acid sequence of SEQ ID NO: 65; and A polypeptide (III) consisting of the amino acid sequence of SEQ ID NO: 66.

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