Interleukin 2 (il-2) and CD123 NK cell engager combination therapy

Abstract

The present disclosure relates to methods of treating cancer with the combination of a binding protein comprising a first and a second antigen binding domain (ABD), wherein the first ABD binds specifically to human CD123 and the second ABD binds specifically to human NKp46, and an interleukin 2 (IL-2) polypeptide.

Description

[0001] PAT24089-WO-PCT

[0002] 768838: SA9-904PC

[0003] INTERLEUKIN 2 (IL-2) AND CD123 NK CELL ENGAGER COMBINATION THERAPY

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit of European Priority Application No. 24306438.3, filed September 4, 2024, which is incorporated by reference in its entirety for all purposes.

[0006] BACKGROUND

[0007] Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are heterogeneous clonal neoplastic diseases, which are thought to arise from subpopulations of leukemic stem cells, which tend to be resistant to conventional chemotherapy, and which may be further responsible for disease relapse.

[0008] CD 123 -targeting natural killer (NK) cell engaging binding proteins (CD123 NKCE) are an efficient class of therapeutics for treating AML and MDS. However, there exists a need for enhanced combination therapies for these diseases.

[0009] SUMMARY

[0010] In one aspect, the disclosure provides a method of treating cancer in a subject, comprising administering to the subject: i) a binding protein comprising a first and a second antigen binding domain (ABD), wherein the first ABD binds specifically to human CD 123 and the second ABD binds specifically to human NKp46; and ii) an interleukin-2 (IL-2) polypeptide or variant thereof, thereby treating the cancer in the subj ect.

[0011] In some embodiments, each of said ABD comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3); and wherein: (i) the first ABD comprises: - a VH1 comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 1 to 3 respectively or corresponding to the amino acid sequences of SEQ ID NO: 4 to 6 respectively, and - a VL1 comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 7 to 9 respectively or corresponding to the amino acid sequences of SEQ ID NO: 10 to 12 respectively; and (ii) the second ABD comprises: - a VH2 comprising a CDR-H1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 13 to 15 respectively; the amino acid sequences of SEQ ID NO: 16 to 18 respectively; the amino acid sequences of SEQ ID NO: 19 to 21 respectively; the amino acid sequences of SEQ ID NO: 22 to 24 respectively; or the amino acid sequences of SEQ ID NO: 16, 25 and 26 respectively; and - a VL2 comprising a CDR-L1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 27 to 29 respectively; the amino acid sequences of SEQ ID NO: 30 to 32 respectively; the amino acid sequences of SEQ ID NO: 33 to 35 respectively; the amino acid sequences of SEQ ID NO: 36 to 38 respectively; or the amino acid sequences SEQ ID NO: 39, 31 and 40 respectively.

[0012] In some embodiments, each of said ABD comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3); and wherein: (i) the first ABD comprises: - a VH1 comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 88 to 90 respectively, and - a VL1 comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 94 to 96 respectively; and (ii) the second ABD comprises: - a VH2 comprising a CDR-H1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 13 to 15 respectively; the amino acid sequences of SEQ ID NO: 16 to 18 respectively; the amino acid sequences of SEQ ID NO: 19 to 21 respectively; the amino acid sequences of SEQ ID NO: 22 to 24 respectively; orthe amino acid sequences of SEQ ID NO: 16, 25 and 26 respectively; and - a VL2 comprising a CDR-L1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 27 to 29 respectively; the amino acid sequences of SEQ ID NO: 30 to 32 respectively; the amino acid sequences of SEQ ID NO: 33 to 35 respectively; the amino acid sequences of SEQ ID NO: 36 to 38 respectively; or the amino acid sequences SEQ ID NO: 39, 31 and 40 respectively.

[0013] In some embodiments, each of said ABD comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3); and wherein: (i) the first ABD comprises: - a VH1 comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 91 to 93 respectively, and - a VL1 comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 97 to 99 respectively; and (ii) the second ABD comprises: - a VH2 comprising a CDR-H1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 13 to 15 respectively; the amino acid sequences of SEQ ID NO: 16 to 18 respectively; the amino acid sequences of SEQ ID NO: 19 to 21 respectively; the amino acid sequences of SEQ ID NO: 22 to 24 respectively; orthe amino acid sequences of SEQ ID NO: 16, 25 and 26 respectively; and - a VL2 comprising a CDR-L1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 27 to 29 respectively; the amino acid sequences of SEQ ID NO: 30 to 32 respectively; the amino acid sequences of SEQ ID NO: 33 to 35 respectively; the amino acid sequences of SEQ ID NO: 36 to 38 respectively; or the amino acid sequences SEQ ID NO: 39, 31 and 40 respectively.

[0014] In some embodiments, the binding protein comprises all or part of an immunoglobulin Fc region or variant thereof. In some embodiments, all or part of the immunoglobulin Fc region or variant thereof binds to a human Fc-y receptor.

[0015] In some embodiments, the binding protein comprises three polypeptide chains (I), (II) and (III) that form two ABDs, as defined below: (I): light chain variable domain 1A (VIA) - light chain constant domain 1 A (CIA) - Hingei - (CH2-CH3)A; (II): heavy chain variable domain IB (VIB) - heavy chain constant domain IB (CIB) - Hinge? - (CH2-CH3)B - LI - heavy chain variable domain 2 A (V?A) - heavy chain constant domain 2 A (C?A) - Hinge?; and (III): light chain variable domain 2B (V?B) - light chain constant domain 2B (C?B), wherein: VIA and VIB form a binding pair Vi (VHI / VLI); V?A and V?B form a binding pair V? (VH? / VL2); CIA and Cm form a pair Ci (CH1 / CL) and C?A and C?B form a pair C2 (CH1 / CL) wherein CH I is an immunoglobulin heavy chain constant domain 1 and CL is an immunoglobulin light chain constant domain; Hingei, Hinge? and Hinge? are identical or different and correspond to all or part of an immunoglobulin hinge region; (CH2-CH3)A and (CH2-CH3)B are identical or different, and comprise an immunoglobulin heavy chain constant domain 2 (CH2) and an immunoglobulin heavy chain constant domain 3 (CH3); LI is an amino acid linker. In some embodiments, VIA is VLI and VIB is VHI . In some embodiments, V?A is VH2 and V?B is VL2.

[0016] In some embodiments, (a) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR- L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 27; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 28; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29; (b) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32; (c) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34; a CDR- L3 comprising the amino acid sequence of SEQ ID NO: 35; (d) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; VL2 comprises a CDR- L1 comprising the amino acid sequence of SEQ ID NO: 36; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38; (e) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 39; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40; (f) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR- L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 27; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 28; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29; (g) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32; (h) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34; a CDR- L3 comprising the amino acid sequence of SEQ ID NO: 35; (i) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; VL2 comprises a CDR- L1 comprising the amino acid sequence of SEQ ID NO: 36; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38; or (j) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 39; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0017] In some embodiments, (a) VHI and VLI corresponds to the amino acid sequences of SEQ ID NO: 41 and 43 respectively or corresponds to the amino acid sequences of SEQ ID NO: 42 and 44 respectively; and / or (b) VH2 and VL2 corresponds 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; the amino acid sequences of SEQ ID NO: 51 and 59 respectively; or the amino acid sequences of SEQ ID NO: 52 and 60 respectively.

[0018] In some embodiments: (a) VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 45; VL2 comprises the amino acid sequence of SEQ ID NO: 53; (b) VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 46; VL2 comprises the amino acid sequence of SEQ ID NO: 54; (c) VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 47; VL2 comprises the amino acid sequence of SEQ ID NO: 55; (d) VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 48; VL2 comprises the amino acid sequence of SEQ ID NO: 56; (e) VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 49; VL2 comprises the amino acid sequence of SEQ ID NO: 57; (f) VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 50; VL2 comprises the amino acid sequence of SEQ ID NO: 58; (g) VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 51; VL2 comprises the amino acid sequence of SEQ ID NO: 59; (h) VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 52; VL2 comprises the amino acid sequence of SEQ ID NO: 60; (i) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 45; VL2 comprises the amino acid sequence of SEQ ID NO: 53; (j) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 46; VL2 comprises the amino acid sequence of SEQ ID NO: 54; (k) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 47; VL2 comprises the amino acid sequence of SEQ ID NO: 55; (1) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 48; VL2 comprises the amino acid sequence of SEQ ID NO: 56; (m) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 49; VL2 comprises the amino acid sequence of SEQ ID NO: 57; (n) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 50; VL2 comprises the amino acid sequence of SEQ ID NO: 58; (o) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 51; VL2 comprises the amino acid sequence of SEQ ID NO: 59; (p) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 52; VL2 comprises the amino acid sequence of SEQ ID NO: 60.

[0019] In some embodiments: polypeptide (I) comprises an amino acid sequence of SEQ ID NO: 64; polypeptide (II) comprises an amino acid sequence of SEQ ID NO: 65; and polypeptide (III) comprises an amino acid sequence of SEQ ID NO: 66.

[0020] In some embodiments: polypeptide (I) consists of an amino acid sequence of SEQ ID NO: 64; polypeptide (II) consists of an amino acid sequence of SEQ ID NO: 65; and polypeptide (III) consists of an amino acid sequence of SEQ ID NO: 66.

[0021] In some embodiments, the IL-2 polypeptide comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 84.

[0022] In some embodiments, the IL-2 polypeptide comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 85.

[0023] In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 85 in which at least one amino acid residue is replaced by an unnatural amino acid. In some embodiments, the position of the at least one unnatural amino acid is selected from K34, T36, R37, T40, F41, K42, F43, Y44, E59, E60, E61, K63, P64, E67, V68, N70, L71, M103, C104, and Y107, wherein the residue positions correspond to the positions 34, 36, 37, 40, 41, 42, 43, 44, 60, 61, 63, 64, 67, 68, 70, 71, 103, 104, and 106 as set forth in SEQ ID NO: 85. In some embodiments, the position of the at least one unnatural amino acid is selected from T36, R37, T40, F41, K42, F43, Y44, E60, E61, P64, E67, and L71 , wherein the residue positions correspond to the positions 36, 37, 40, 41, 42, 43, 44, 60, 61, 64, 67, and 71 as set forth in SEQ ID NO: 85. In some embodiments, the position of the at least one unnatural amino acid is selected from K34, K63, V68, N70, M103, C104, and Y106, wherein the residue positions correspond to the positions 34, 63, 68, 70, 103, 104, and 106 as set forth in SEQ ID NO: 85. In some embodiments, the position of the at least one unnatural amino acid is selected from T36, R37, T40, Y44, E60, E67, and L71, wherein the residue positions correspond to the positions 36, 37, 40, 44, 60, 67, and 71 as set forth in SEQ ID NO: 85.

[0024] In some embodiments, the position of the at least one unnatural amino acid is selected from F41, K42, F43, E61, and P64, wherein the residue positions correspond to the positions 41, 42, 43, 61, and 64 as set forth in SEQ ID NO: 85. In some embodiments, the at least one unnatural amino acid: is a lysine analogue; comprises an aromatic side chain; comprises an azido group; comprises an alkyne group; or comprises an aldehyde or ketone group.

[0025] In some embodiments, the at least one unnatural amino acid does not comprise an aromatic side chain.

[0026] In some embodiments, the at least one unnatural amino acid comprises N6-azidoethoxy- L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid, 2- amino-8-oxooctanoic acid, p- acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p- propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L- phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, 0-4-allyl-L-tyrosine, 4- propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3- oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic, or selenocysteine.

[0027] In some embodiments, the IL-2 polypeptide is covalently attached to a conjugating moiety through the at least one unnatural amino acid.

[0028] In some embodiments, the conjugating moiety comprises a water-soluble polymer, a lipid, a protein, or a peptide.

[0029] In some embodiments, the water-soluble polymer comprises polyethylene glycol (PEG), polypropylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N- acryloylmorpholine), or a combination thereof.

[0030] In some embodiments, the water-soluble polymer comprises a PEG molecule. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG.

[0031] In some embodiments, the conjugating moiety is indirectly bound to the at least one unnatural amino acid of the IL-2 polypeptide through a linker. In some embodiments, the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or a non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof.

[0032] In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion, a C- terminal deletion, or a combination thereof.

[0033] In some embodiments, the N-terminal deletion comprises a deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from the N-terminus, wherein the residue positions are in reference to the positions in SEQ ID NO: 85. In some embodiments, the C-terminal deletion comprises a deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues from the C-terminus, wherein the residue positions are in reference to the positions in SEQ ID NO: 85.

[0034] In some embodiments, the conjugating moiety or the unnatural amino acid impairs or blocks the binding of IL-2 with IL-2Ra.

[0035] In some embodiments, the IL-2 polypeptide comprises an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 85, wherein the amino acid at position P64 is replaced by the structure of Formula

[0036] Formula (I) wherein:

[0037]

[0038] W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; q is 1, 2, or 3;

[0039] X has the structure: wherein X-l indicates the point of attachment to the preceding amino acid residue; and wherein X+l indicates the point of attachment to the following amino acid residue.

[0040] In some embodiments, the PEG group has an average molecular weight of about 30 kDa.

[0041] In some embodiments, the

[0042] In some embodiments, the

[0043] In some embodiments, the

[0044] In some embodiments, the IL-2 conjugate comprises (i) an IL-2 conjugate in which the structure of Formula (I) has the structure of Formula (IV), and / or (ii) an IL-2 conjugate in which the structure of Formula (I) has the structure of or Formula (V):

[0045] Formula (V); wherein: q is 1, 2, or 3;

[0046] X has the structure: wherein X-l indicates the point of attachment to the preceding amino acid residue; and wherein X+l indicates the point of attachment to the following amino acid residue.

[0047] In some embodiments, the IL-2 conjugate comprises (i) an IL-2 conjugate in which the structure of Formula (I) has the structure of Formula (XII), and / or (ii) an IL-2 conjugate in which the structure of Formula (I) has the structure of or Formula (XIII):

[0048] Formula (XII);

[0049] Formula (XIII); wherein: n is an integer such that a PEG group having the structure of -(OCEECH^n-OCHa has a molecular weight of about 30 kDa; q is 1, 2, or 3; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 85 that are not replaced.

[0050] In some embodiments, the q is 1. In some embodiments, the q is 2. In some embodiments, the q is 3.

[0051] In some embodiments, the IL-2 polypeptide that is covalently attached to a conjugating moiety has reduced binding between the IL-2 polypeptide and interleukin 2 receptor alpha (IL- 2Ra) but retains significant binding with interleukin 2 Py receptor (IL-2R Py) signaling complex to form an IL-2 / IL- 2RPy complex, wherein the reduced binding to IL-2Ra is compared to binding between a wild-type IL-2 polypeptide and IL-2Ra.

[0052] In some embodiments, the reduced binding is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% reduced binding to IL-2Ra relative to a wild-type IL-2 polypeptide. In some embodiments, the reduced binding is about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, or more reduced binding to IL-2Ra relative to a wild-type IL-2 polypeptide.

[0053] In some embodiments, the binding protein is administered to the subject prior to the IL- 2 polypeptide. In some embodiments, the IL-2 polypeptide is administered to the subject prior to the binding protein. In some embodiments, the binding protein and the IL-2 polypeptide thereof are administered contemporaneously to the subject.

[0054] In some embodiments, the cancer is a blood cancer.

[0055] In some embodiments, the cancer is myelodysplastic syndrome (MDS) or a lymphoproliferative disorder.

[0056] In some embodiments, the cancer is Acute Myeloid Leukemia (AML). In some embodiments, cytotoxicity of cancer cells in the subject is induced after administration of the binding protein and the IL-2 polypeptide. In some embodiments, NK cell activation in the subject is induced after administration of the binding protein and the IL-2 polypeptide. In some embodiments, NK cell activation is determined by measuring CD69 expression from NK cells in the subject. In some embodiments, administration of the binding protein and the IL-2 polypeptide to the subject results in increased release of one or more effector cytokines. In some embodiments, the one or more effector cytokines are IFN-y, TNF- a, MIP-la, MIP-ip, Granzyme A, Granzyme B, and Lymphotactin.

[0057] BRIEF DESCRIPTION OF THE FIGURES

[0058] FIG. 1. Schematic representation of the F25 format, including one human NKp46 binding site and one human CD123 binding site. The C-terminus of the polypeptide is on the left-hand side and the N-terminus is on the right-hand side.

[0059] FIG. 2A - FIG. 2E show two-dimensional schematic representations of the F25, F5, F26 and F6 formats respectively including the relevant domains for each polypeptide chain. In the FIG. 2A to FIG. 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 side. The human CD123 binding domain is formed by the VH / VL pair on the right side.

[0060] FIG. 2A shows a two-dimensional schematic representation of the F25 format. This representation represents the claimed “NKp46-CD123_F25” binding protein.

[0061] FIG. 2B shows a two-dimensional schematic representation of the F5 format. When compared to F25, the F5 differs in that the CL and CH pair of the NKp46 binding domain are swapped, with the third polypeptide chain comprising a CHI domain and a VL domain.

[0062] FIG. 2C shows a two-dimensional schematic representation of the F26 format. This F26 differs from F25 of figure 2A in that it includes a Fc-silent N297S mutation on each CH2 domain.

[0063] FIG. 2D shows a two-dimensional schematic representation of the F6 format. This F6 differs from F5 of figure 2B in that it includes a Fc-silent N297S mutation on each CH2 domain.

[0064] FIG. 2E shows a two-dimensional detailed representation of a variant of the F25 format. This F25 format representation corresponds to the one in Figure 2A. FIG. 3 shows a proposed mechanism of action of a Natural Killer Cell Engager (NKCE) for killing, following joint binding of a tumor cell (i.e. an AML cell line; i.e. MOLM- 13) expressing CD123 and a natural killer (NK) cell expressing NKp46 and a Fey receptor (CD16a). Reproduced and adapted from Gauthier, L. et al. (“Multifunctional natural killer cell engagers targeting NKp46 trigger protective tumor immunity”. Cell 177, 1701-1713 (2019)).

[0065] FIG. 4A shows a graph depicting growth curves of THP-1 GFP cells alone (black curve) or in presence of healthy donor (HD) NK cells + isotype control (IC) at 1 ng / ml (light blue curve), HD NK cells + IC at 1 ng / ml + pegenzileukin (designated “IL2” in graph) at 100 ng / ml (blue curve), HD NK cells + CD123 NKCE (designated “CD123” in graph) at 1 ng / ml (light red curve), and HD NK cells + CD123 NKCE at 1 ng / ml in combination with pegenzileukin at 100 ng / ml (red curve). E:T ratio = 1 : 1. Representative of n=4 NK healthy donors. Black arrows indicate which legend symbol corresponds with a particular line of data in the graph.

[0066] FIG. 4B shows a graph depicting growth curves of THP-1 GFP cells alone (black curve) or in presence of HD NK cells + IC at 10 ng / ml (light blue curve), HD NK cells + IC at 10 ng / ml + pegenzileukin at 100 ng / ml (blue curve), HD NK cells + CD123 NKCE at 10 ng / ml (light red curve), and HD NK cells + CD123 NKCE at 10 ng / ml in combination with pegenzileukin at 100 ng / ml (red curve). E:T ratio = 1 : 1. Representative of n=4 NK healthy donors. Black arrows indicate which legend symbol corresponds with a particular line of data in the graph.

[0067] FIG. 5 shows a plot of cytotoxic activity of the CD 123 NKCE in combination with pegenzileukin against THP-1 tumor cells in presence of HD NK cells. Boxplots of each compound at each dose. ***: p<0.001 and **: p<0.01 obtained comparing each single compound to combination CD 123 NKCE + pegenzileukin at each dose with a mixed model on AUC followed by Dunnett’s test. The dose of 100 ng / mL was not considered for the statistical analysis but is still presented in FIG. 5. PP29 and PP30 represent different assays. DI, D2, D3, and D4 represent different NK cell donors.

[0068] FIG. 6A shows a graph depicting growth curves of MOLM-13 RFP cells alone (black curve) or in presence of HD NK cells + isotype control (IC) at 1 ng / ml (light blue curve), HD NK cells + IC at 1 ng / ml + pegenzileukin at 100 ng / ml (blue curve), HD NK cells + CD123 NKCE at 1 ng / ml (light red curve), and HD NK cells + CD 123 NKCE at 1 ng / ml in combination with pegenzileukin at 100 ng / ml (red curve). E:T ratio = 1 : 1. Representative of n=4 NK healthy donors. Black arrows indicate which legend symbol corresponds with a particular line of data in the graph.

[0069] FIG. 6B shows a graph growth curves of MOLM-13 RFP cells alone (black curve) or in presence of HD NK cells + IC at 10 ng / ml (light blue curve), HD NK cells + IC at 10 ng / ml + pegenzileukin at 100 ng / ml (blue curve), HD NK cells + CD 123 NKCE at 10 ng / ml (light red curve), and HD NK cells + CD123 NKCE at 10 ng / ml in combination with pegenzileukin at 100 ng / ml (red curve). E:T ratio = 1 : 1. Representative of n=4 NK healthy donors. Black arrows indicate which legend symbol corresponds with a particular line of data in the graph.

[0070] FIG. 6C shows a graph growth curves of MOLM-13 RFP cells alone (black curve) or in presence of HD NK cells + IC at 100 ng / ml (light blue curve), HD NK cells + IC at 100 ng / ml + pegenzileukin at 100 ng / ml (blue curve), HD NK cells + CD123 NKCE at 100 ng / ml (light red curve), and HD NK cells + CD123 NKCE at 100 ng / ml in combination with pegenzileukin at 100 ng / ml (red curve). E:T ratio = 1 : 1. Representative of n=4 NK healthy donors. Black arrows indicate which legend symbol corresponds with a particular line of data in the graph.

[0071] FIG. 7 shows a plot of cytotoxic activity of the CD 123 NKCE in combination with pegenzileukin against MOLM-13 tumor cells in presence of HD NK cells. Boxplots of each compound at each dose. ***: p<0.001 obtained comparing each single compound to combination CD 123 NKCE + pegenzileukin at each dose with a mixed model on AUC followed by Dunnett’s test. PP48 and PP50 represent different assays. D5, D6, D7, and D8 represent different NK cell donors. E:T ratio = 1 :1.

[0072] FIG. 8A shows a graph depicting growth curves of MOLM-13 RFP cells alone (black curve) or in presence of HD NK cells + isotype control (IC) at 10 ng / ml (light blue curve), HD NK cells + IC at 10 ng / ml + pegenzileukin at 100 ng / ml (blue curve), HD NK cells + CD123 NKCE at 10 ng / ml (light red curve), and HD NK cells + CD123 NKCE at 10 ng / ml in combination with pegenzileukin at 100 ng / ml (red curve). E:T ratio = 1 :2. Representative of n=3 NK healthy donors. Black arrows indicate which legend symbol corresponds with a particular line of data in the graph.

[0073] FIG. 8B shows a graph depicting growth curves of MOLM-13 RFP cells alone (black curve) or in presence of HD NK cells + IC at 100 ng / ml (light blue curve), HD NK cells + IC at 100 ng / ml + pegenzileukin at 100 ng / ml (blue curve), HD NK cells + CD123 NKCE at 100 ng / ml (light red curve), and HD NK cells + CD123 NKCE at 100 ng / ml in combination with pegenzileukin at 100 ng / ml (red curve). E:T ratio = 1 :2. Representative of n=3 NK healthy donors. Black arrows indicate which legend symbol corresponds with a particular line of data in the graph.

[0074] FIG. 8C shows a plot of cytotoxic activity of the CD 123 NKCE in combination with pegenzileukin against MOLM-13 tumor cells in presence of HD NK cells. Boxplots of each compound at each dose. ***: p<0.001 obtained comparing each single compound to combination CD 123 NKCE + pegenzileukin at each dose with a mixed model on AUC followed by Dunnett’s test. PP50 and PP51 represent different assays. D7, D8, and D9 represent different NK cell donors. E:T ratio of 1 :2.

[0075] FIG. 9 shows a plot of significant differences in CD69 expression as a percentage of CD69-positive NK cells and the flow cytometry Median Fluorescent Intensity (MFI) of CD69 on NK cells. The numbers 10, 100, and 1000 in the plot represent the concentration of the CD123 NKCE or pegenzileukin in ng / ml. MOLM-13 cells were incubated with NK cells and an isotype control, the CD 123 NKCE, and / or pegenzileukin for 24 hours followed by CD69 expression determination. #1 - NK cells (NK) + M0LM13 cells (M0LM13) + isotype control (IC) 1000 + pegenzileukin (IL2) compared to NK + M0LM13 + IC; #2 - NK + M0LM13 + IC 100 + IL2 compared to NK + M0LM13 + IC 100; #3 - NK + M0LM13 + CD123 NKCE 1000 + IL2 compared to NK + M0LM13 + CD123 NKCE 1000; #4 - NK + M0LM13 + CD 123 NKCE 100 + IL2 compared to NK + M0LM13 + CD 123 NKCE 100; #5 - NK + M0LM13 + CD123 NKCE 10 + IL2 compared to NK + M0LM13 + CD123 NKCE 10; #6 - NK + M0LM13 + CD123 NKCE 1000 + IL2 compared to NK + M0LM13 + IL2; #7 - NK + M0LM13 + CD123 NKCE 100 + IL2 compared to NK + M0LM13 + IL2; #8 - NK + M0LM13 + CD 123 NKCE 10 + IL2 compared to NK + M0LM13 + IL2; #9 - NK + M0LM13 + CD123 NKCE 1000 + IL2 compared to NK + M0LM13 + IC 1000; #10 - NK + M0LM13 + CD123 NKCE 100 + IL2 compared to NK + M0LM13 + IC 100; #11 - NK + M0LM13 + CD123 NKCE 10 + IL2 compared to NK + M0LM13 + IC 100; #12 - NK + M0LM13 compared to NK + M0LM13 + IC 1000; #13 - NK + M0LM13 compared to NK + M0LM13 + IC 100; #14 - NK + M0LM13 compared to NK alone.

[0076] FIG. 10 shows boxplots of each compound for normalized CD69 expression (MFI) on a log scale. NK38, NK59, NK 64, and NK92 represent different NK cell donors.

[0077] FIG. 11 shows boxplots of each compound for normalized CD69 expression (%) on a log scale. NK38, NK59, NK 64, and NK92 represent different NK cell donors. FIG. 12 shows a histogram of CD69 expression in MFI and percentage. ¥¥¥¥p<0.0001 CD123 NKCE (“CD123” in figure) vs. IC at corresponding concentrations (CD123 NKCE 10 ng / ml compared to IC 100 ng / ml); §§§§p<0.0001 pegenzileukin / IC vs. IC at corresponding concentrations; ****p<0.0001 CD123 NKCE / pegenzileukin vs. CD123 NKCE at corresponding concentrations; ####p<0.0001, ###p<0.001, ##p<0.01 CD123 NKCE / pegenzileukin vs. pegenzileukin / IC at corresponding concentrations; p-values are obtained on log-transformed normalized data for CD69 (MFI) and on normalized data for CD69 (%) whereas graphs are on raw data. Pegenzileukin was used at 100 ng / ml. Values are expressed as geometric mean * GSD for CD69 (MFI) and mean ± SD for CD69 (%); N= 4 healthy donor- derived NK cells. For each pair of bars in the graph, the left bar is without pegenzileukin and the right bar is with pegenzileukin.

[0078] FIG. 13 shows a summary of significant differences (adjusted p value) in cytokine / chemokine concentrations at 24 hours and 48 hours under the indicated conditions. #1 - NK cells (NK) + M0LM13 cells (M0LM13) + CD123 1000 + pegenzileukin compared to NK + M0LM13 + IC 1000 + pegenzileukin; #2 -NK + M0LM13 + CD123 1000 + pegenzileukin compared to NK + M0LM13 + CD123 1000; #3 -NK + M0LM13 + CD123 100 + pegenzileukin compared to NK + M0LM13 + IC 100 + pegenzileukin; #4 -NK + M0LM13 + CD123 100 + pegenzileukin compared to NK + M0LM13 + CD 123 100; #5 -NK + M0LM13 + CD123 10 + pegenzileukin compared to NK + M0LM13 + IC 100 + pegenzileukin; #6 -NK + M0LM13 + CD 123 10 + pegenzileukin compared to NK + M0LM 13 + CD123 10; #7 -NK + M0LM13 + IC 1000 + pegenzileukin compared to NK + M0LM13 + IC 1000; #8 -NK + M0LM13 + IC 100 + pegenzileukin compared to NK + M0LM13 + IC 100; #9 -NK + M0LM13 + CD123 1000 compared to NK + M0LM13 + IC 1000; #10 -NK + M0LM13 + CD123 100 compared to NK + M0LM13 + IC 100; #11 -NK + M0LM13 + CD 123 10 compared to NK + M0LM13 + IC 10; #12 -NK + M0LM13 compared to NK + M0LM13 + IC 1000; #13 -NK + M0LM13 compared to NK + M0LM13 + IC 100; #13 -NK + M0LM13 compared to NK alone.

[0079] FIG. 14 shows boxplots of each compound for IFN-y concentration on log scale. NK38, NK59, NK 64, and NK92 represent different NK cell donors.

[0080] FIG. 15 shows a histogram for IFN-y at 24 hours and 48 hours. ¥¥¥¥p<0.0001, ¥¥¥p<0.00 l , ¥¥p<0.0 l and ¥p<0.05 CD123 NKCE vs. IC at corresponding concentrations (CD123 NKCE lOng / ml compared to IC 100 ng / ml); §§§§p<0.0001 pegenzileukin / IC vs. IC at corresponding concentrations; ****p<0.0001 CD123 NKCE / pegenzileukin vs. CD123 NKCE at corresponding concentrations; ####p<0.0001, ##p<0.01 and #p<0.05 CD123 NKCE / pegenzileukin vs. pegenzileukin / IC at corresponding concentrations; p-values are obtained on normalized data whereas graphs are on raw data, pegenzileukin was used at 100 ng / ml. Values are expressed as mean ± SD; N= 4 healthy donor-derived NK cells. For each pair of bars in the graph, the left bar is without pegenzileukin and the right bar is with pegenzileukin.

[0081] FIG. 16 shows boxplots of each compound for TNF-a concentration on log scale. NK38, NK59, NK 64, and NK92 represent different NK cell donors.

[0082] FIG. 17 shows a histogram for TNF-a at 24 hours and 48 hours. ¥¥¥¥p<0.0001 and ¥p<0.05 CD123 NKCE vs. IC at corresponding concentrations (CD123 NKCE lOng / ml compared to IC 100 ng / ml); §§§§p<0.0001 pegenzileukin / IC vs. IC at corresponding concentrations; ****p<0.0001 and **p<0.01 CD123 NKCE / pegenzileukin vs. CD123 NKCE at corresponding concentrations; ####p<0.0001 CD123 NKCE / pegenzileukin vs. pegenzileukin / IC at corresponding concentrations; p-values are obtained on normalized data whereas graphs are on raw data, pegenzileukin always at 100 ng / ml. Values are expressed as mean ± SD; N= 4 healthy donor-derived NK cells. For each pair of bars in the graph, the left bar is without pegenzileukin and the right bar is with pegenzileukin.

[0083] FIG. 18 shows boxplots of each compound for MIP-la concentration on log scale. NK38, NK59, NK 64, and NK92 represent different NK cell donors.

[0084] FIG. 19 shows a histogram for MIP-la at 24 hours and 48 hours. ¥¥¥¥p<0.0001 and ¥¥p<0.0 l CD123 NKCE vs. IC at corresponding concentrations (CD123 NKCE lOng / ml compared to IC 100 ng / ml); §§§§p<0.0001 pegenzileukin / IC vs. IC at corresponding concentrations; ****p<0.0001, ***p<0.001 and **p<0.01 CD123 NKCE / pegenzileukin vs. CD123 NKCE at corresponding concentrations; ####p<0.0001 and ###p<0.001 CD123 NKCE / pegenzileukin vs. pegenzileukin / IC at corresponding concentrations; p-values are obtained on normalized data whereas graphs are on raw data, pegenzileukin always at 100 ng / ml. Values are expressed as mean ± SD; N= 4 healthy donor-derived NK cells. For each pair of bars in the graph, the left bar is without pegenzileukin and the right bar is with pegenzileukin.

[0085] FIG. 20 shows boxplots of each compound for MIP-ip concentration on log scale. NK38, NK59, NK 64, and NK92 represent different NK cell donors.

[0086] FIG. 21 shows a histogram for MIP-ip at 24 hours and 48 hours. ¥¥¥p<0.00 l and ¥¥p<0.0 l CD123 NKCE vs. IC at corresponding concentrations (CD123 NKCE lOng / ml compared to IC 100 ng / ml); §§§§p<0.0001 pegenzileukin / IC vs. IC at corresponding concentrations; ****p<0.0001, ***p<0.001 CD123 NKCE / pegenzileukin vs. CD123 NKCE at corresponding concentrations; ###p<0.001 and ##p<0.01 CD123 NKCE / pegenzileukin vs. pegenzileukin / IC at corresponding concentrations; p-values are obtained on normalized data whereas graphs are on raw data, pegenzileukin always at 100 ng / ml. Values are expressed as mean ± SD; N= 4 healthy donor-derived NK cells. For each pair of bars in the graph, the left bar is without pegenzileukin and the right bar is with pegenzileukin.

[0087] FIG. 22 shows boxplots of each compound for Granzyme A concentration on log scale. NK38, NK59, NK 64, and NK92 represent different NK cell donors.

[0088] FIG. 23 shows a histogram for Granzyme A at 24 hours and 48 hours. ¥¥¥¥p<0.0001 and ¥p<0.05 CD123 NKCE vs. IC at corresponding concentrations (CD123 NKCE lOng / ml compared to IC 100 ng / ml); §§§§p<0.0001 pegenzileukin / IC vs. IC at corresponding concentrations; ****p<0.0001 and ***p<0.001 CD123 NKCE / pegenzileukin vs. CD123 NKCE at corresponding concentrations; #p<0.05 CD123 NKCE / pegenzileukin vs. pegenzileukin / IC at corresponding concentrations; p-values are obtained on normalized data whereas graphs are on raw data, pegenzileukin always at 100 ng / ml. Values are expressed as mean ± SD; N= 4 healthy donor-derived NK cells. For each pair of bars in the graph, the left bar is without pegenzileukin and the right bar is with pegenzileukin.

[0089] FIG. 24 shows boxplots of each compound for Granzyme B concentration on log scale. NK38, NK59, NK 64, and NK92 represent different NK cell donors.

[0090] FIG. 25 shows a histogram for Granzyme B at 24 hours and 48 hours. ¥p<0.05 CD 123 NKCE vs. IC at corresponding concentrations (CD 123 NKCE lOng / ml compared to IC 100 ng / ml); §§§§p<0.0001 pegenzileukin / IC vs. IC at corresponding concentrations; ****p<0.0001 and ***p<0.001 CD123 NKCE / pegenzileukin vs. CD123 NKCE at corresponding concentrations; p-values are obtained on normalized data whereas graphs are on raw data, pegenzileukin always at 100 ng / ml. Values are expressed as mean ± SD; N= 4 healthy donor-derived NK cells. For each pair of bars in the graph, the left bar is without pegenzileukin and the right bar is with pegenzileukin.

[0091] FIG. 26 shows boxplots of each compound for Lymphotactin concentration on log scale. NK38, NK59, NK 64, and NK92 represent different NK cell donors.

[0092] FIG. 27 shows a histogram for Lymphotactin at 24 hours and 48 hours. ¥¥p<0.0 l and ¥p<0.05 CD123 NKCE vs. IC at corresponding concentrations (CD123 NKCE lOng / ml compared to IC 100 ng / ml); §§§§p<0.0001, §§§p<0.001 pegenzileukin / IC vs. IC at corresponding concentrations; ****p<0.001, ***p<0.001 and **p<0.01 CD123 NKCE / pegenzileukin vs. CD123 NKCE at corresponding concentrations; ####p<0.0001, ##p<0.01 CD123 NKCE / pegenzileukin vs. pegenzileukin / IC at corresponding concentrations; p-values are obtained on normalized data whereas graphs are on raw data, pegenzileukin always at 100 ng / ml. Values are expressed as mean ± SD; N= 4 healthy donor-derived NK cells. For each pair of bars in the graph, the left bar is without pegenzileukin and the right bar is with pegenzileukin.

[0093] DETAILED DESCRIPTION

[0094] The disclosure provides methods of treating cancer with the combination of binding proteins that bind NKp46 on NK cells and CD 123 on cancer cells (CD 123 NK cell engagers (NKCEs)) and interleukin-2 (IL-2) polypeptides and IL-2 conjugates thereof. The combination of the CD123 NKCEs of the disclosure and the IL-2 polypeptides and IL-2 conjugates thereof of the disclosure enhance cancer cell cytotoxicity. For example, the combination of a CD123 NKCE and an exemplary IL-2 polypeptide conjugate pegenzileukin displayed enhanced in vitro cytotoxicity against acute myeloid leukemia (AML) cell lines MOLM-13 and THP-1. Moreover, said combination of a CD123 NKCE and an exemplary IL- 2 polypeptide conjugate pegenzileukin induced NK cell activation (as determined by CD69 expression) and enhanced effector cytokine and chemokine release from healthy donor-derived NK cells in presence of MOLM-13 AML tumor cells.

[0095] Definitions

[0096] As used herein, the “CD123” marker, or “Cluster of Differentiation 123” is also known as “Interleukin 3 receptor, alpha (IL3RA)” or “IL3R”, “IL3RX”, “IL3RY”, “IL3RAY”, “hlL-3Ra” and denotes an interleukin 3 specific subunit of a heterodimeric cytokine receptor. The functional interleukin 3 receptor is a heterodimer that comprises a specific alpha chain (IL- SA; CD123) and the IL-3 receptor beta chain (P9; CD131) that is shared with the receptors for granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 5 (IL-5). CD123 is a type I integral transmembrane protein with a deduced Molecular Weight of about 43kDa containing an extracellular domain involved in IL-3 binding, a transmembrane domain and a short cytoplasmic tail of about 50 amino acids. The extracellular domain is composed of two regions: a N-terminal region of about 100 amino acids, the sequence of which exhibits similarity to equivalent regions of the GM-CSF and IL-5 receptor alpha-chains; and a region proximal to the transmembrane domain that contains four conserved cysteine residues and a motif, common to other members of this cytokine receptor family. The IL-3 binding domain comprises about 200 amino acid residue cytokine receptor motifs (CRMs) made up of two Ig-like folding domains. The extracellular domain of CD123 is highly glycosylated, with N-glycosylation necessary for both ligand binding and receptor signaling. The protein family gathers three members: IL3RA (CD123A), CSF2RA and IL5RA. The overall structure is well conserved between the three members, but sequence homologies are very low. One 300 amino-acid long isoform of CD123 has been discovered so far, but only on the RNA level which is accessible on the Getentry database under the accession number ACM241 16.1. A reference sequence of full-length human CD123 protein, including signal peptide, is available from the NCBI database under the accession number NP 002174.1 and under the Uniprot accession number P26951.

[0097] The extracellular domain of human CD123 (ECD) consists of the amino acid sequence of SEQ ID NO: 81. CD123 (the interleukin-3 receptor alpha chain IL-3Ra) is a tumor antigen overexpressed in a variety of hematological neoplasms. The majority of AML blasts express surface CD123 and this expression does not vary by subtype of AML. Higher expression of CD123 on AML at diagnosis has been reported to be associated with poorer prognosis.

[0098] CD 123 expression has been reported in other hematological malignancies including myelodysplasia, systemic mastocytosis, blastic plasmacytoid dendritic cell neoplasm (BPDCN), ALL and hairy cell leukemia.

[0099] As used herein, “Natural killer” or “NK cells” refers to a sub-population of lymphocytes that is involved in non-conventional immunity. NK cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including CD 16, CD56 and / or CD57, NKp46 for human NK cells, the absence of the alpha / beta or gamma / delta TCR complex on the cell surface, the ability to bind to and kill cells that fail to express “self’ MHC / HLA antigens by the activation of specific cytolytic machinery, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify NK cells, using methods well known in the art. Any subpopulation of NK cells will also be encompassed by the term NK cells. Within the context herein “active” NK cells designate biologically active NK cells, including NK cells having the capacity of lysing target cells or enhancing the immune function of other cells. NK cells can be obtained by various techniques known in the art, such as isolation from blood samples, cytapheresis, tissue or cell collections, etc. Useful protocols for assays involving NK cells can be found in Natural Killer Cells Protocols (edited by Campbell KS and Colonna M). Human Press, pp. 219-238 (2000).

[0100] As used herein, the “NKp46” marker, or “Natural cytotoxicity triggering receptor 1”, also known as “CD335” or “NKP46” or “NK-p46” or “LY94” refers to a protein - or polypeptide - encoded by the Ncrl gene. A reference sequence of full-length human NKp46 protein is available from the NCBI database under the accession number NP 004820. The human NKp46 extracellular domain (ECD) corresponds to the amino acid sequence of SEQ ID NO: 80. The human NKp46 mRNA sequence is described in NCBI accession number NM_004829.

[0101] As used herein, the term “Fc-y receptor” or “FcyR” or “Fc-gamma receptor” may refer to both activating and inhibitory FcyRs. Fc-gamma receptors (FcyR) are cellular receptors for the Fc region of an Immunoglobulin G (IgG). Upon binding of complexed IgG, FcyRs can modulate cellular immune effector functions, thereby linking the adaptive and innate immune systems, including ADCC-mediated immune responses. In humans, six classic FcyRs are currently reported: one high-affinity receptor (FcyRI) and five low-to-medium -affinity FcyRs (FcyRIIA, -B and -C, FcyRIIIA and -B). All FcyRs bind the same region on IgG Fc, yet with differing high (FcgRI) and low (FcgRII and FcgRIII) affinities. On a functional level, most of the FcyRs are activating receptors that can induce the cellular responses mentioned above, including ADCC-mediated immune response. Whereas FcyRI, FcyRIIa, FcyRIIc, and FcyRIIIa are activating receptors characterized by an intracellular immunoreceptor tyrosine-based activation motif (TEAM), FcyRIIb has an inhibition motif (ITIM) and is therefore inhibitory. Unless specified otherwise, the term FcyRs encompasses activating receptors, including FcyRI (CD64), FcyRIIA (CD32a), FcyRIIIa (CD16a) and FcyRIIIb (CD16b), and preferably FcyRIIIa (CD 16a).

[0102] As used herein, the term “FcyRIIIa (CD16a)” or “FcyRIIIa” or“CD16a” or“CD16” or “Cluster of Differentiation 16” may refer to a 50-65 kDa cell surface molecule expressed on mast cells, macrophages, and natural killer cells as a transmembrane receptor. FcyRIIIa is an activating receptor containing immunoreceptor tyrosine activating motifs (ITAMs) in the associated FcRy-chain, ITAMs being necessary for receptor expression, surface assembly and signaling. CD 16a is a low affinity receptor for IgG and is an important receptor mediating ADCC (antibody dependent cell mediated cytotoxicity) by NK cells. The high affinity receptor CD 16a is preferentially found on NK cells and monocytes and induces antibody-dependent cellular cytotoxicity (ADCC) upon IgG binding.

[0103] As used herein, the term “FcyRII CD32”, “FcyRII”, “FCGR2” or “CD32a” or “CD32A” or “CD32” or “cluster of differentiation 32” is a surface receptor glycoprotein belonging to the Ig gene superfamily. CD32A is expressed on all myeloid cells but not on lymphocytes. CD32 has a low-affinity for the Fc region of IgG antibodies in monomeric form, but high affinity for IgG immune complexes. CD32 has two major functions: cellular response regulation, and the uptake of immune complexes. Cellular responses regulated by CD32 include phagocytosis, cytokine stimulation, and endocytic transport. Dysregulated CD32 is associated with different forms of autoimmunity, including systemic lupus erythematosus. In humans, there are three major CD32 subtypes: CD32A, CD32B, and CD32C. While CD32A and CD32C are involved in activating cellular responses, CD32B is inhibitory and balances the activating properties of CD32A. CD32A is an activating subtype of CD32 that can be found on a variety of immune cells. Notably, CD32A (FcyRIIA) mediates effector functions of granulocytes, monocytes, B cells, platelets, and dendritic cells upon low affinity binding of aggregated IgG. When bound to an IgG immune complex, the cytosolic ITAM can promote phagocytic activity and cytokine secretion in neutrophils and macrophages.

[0104] As used herein, the term “hFcyRICD64”, “hFcyRI”, or “CD64” or “Cluster of Differentiation 64” is a surface receptor expressed constitutively only on monocytes and macrophages, but is upregulated on granulocytes upon cytokine stimulation.

[0105] As used herein, the terms “Format 5” or “F5”, “Format 25” or “F25”, “Format F6” or “F6” and “Format 26” or “F26” refer to specific binding protein configurations of bispecific or multispecific antibodies specifically designed to engineer multiple antigen binding domains into a single antibody molecule. The multifunctional binding proteins of the present disclosure which comprise a NKp46-binding domain and a CD123-binding domain, are made based on the F25 format, as exemplified in Figure 1 and Figure 2. F25 and format F26 respectively differ from format F5 and F6 in that one CHI / CL pair between the second and third polypeptide chain are swapped to form a CL / CH1 pair. The F5 and F6 format have been previously described in the international patent application WO2017114694, incorporated herein by reference.

[0106] 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 two distinct antigen-binding domains (ABDs). As used herein, the term “specifically binds to” or “binds specifically to” refers to the ability of an antigen-binding domain (ABD) to bind to an antigen (e.g. human NKp46 and / or human CD123) containing an epitope with an Kd of at least about 1 x 10'6M, 1 x 10'7M, 1 x 10'8M, 1 x 10'9M, 1 x IO'10M, 1 x 1 O'11M, 1 x 10'12M, or more, and / or to bind to an epitope with an affinity that is at least twofold greater than its affinity for a nonspecific antigen.

[0107] As used herein, the term “specifically binds to human NK46 polypeptide” may refer to a specific binding toward a polypeptide comprising an amino acid sequence of SEQ ID NO: 80.

[0108] As used herein, the term “specifically to a human CD123 polypeptide” may refer to a specific binding toward a polypeptide comprising an amino acid sequence of SEQ ID NO: 81.

[0109] As used herein, the term “binds to a human Fc-y receptor polypeptide” may refer to a binding toward a polypeptide comprising an amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83.

[0110] 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 “with specificity for” are used interchangeably. Those terms are not construed to refer exclusively to those antibodies, polypeptides and / or multichain polypeptides which actually bind to the recited target / binding partner, but also to those which, although provided in a nonbound form, retain the specificity to the recited target. Binding specificity can be quantitatively determined by an affinity constant KA (or KA) and a dissociation constant KD (or KD).

[0111] As used herein, the term “affinity”, concentration (EC50) or the equilibrium dissociation constant (KD) means the strength of the binding of an antibody or polypeptide to an epitope. The affinity of an antibody is given by a specific type of equilibrium constant, which is the dissociation constant KD, 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 the unbound antibody and [Ag] is the molar concentration of the 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, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of mAbs is the use of surface plasmon resonance (SPR) screening (such as by analysis with a BIAcore™ SPR analytical device). In a non-limitative manner, a KD of less than 50 nM as determined by SPR, and under physiological conditions (e.g. at a pH ranging from 6 to 8 under normal buffer conditions), may generally be considered as indicative of specificity of binding for antigenantigen binding domain (ABD) interactions.

[0112] As an illustration, and according to some particular and exemplified embodiments, binding proteins reported herein comprise:

[0113] - an antigen binding domain which binds specifically to human CD 123 with a KD of less than 10 nM, in particular with a KD of less than 0.5 nM, as determined by SPR, under physiological conditions;

[0114] - an antigen binding domain which binds specifically to human NKp46 with a KD of less than 50 nM, in particular with a KD of less than 20 nM, as determined by SPR, under physiological conditions.

[0115] As used herein, the term “and / or” is a grammatical conjunction that is to be interpreted as encompassing that one or more of the cases it connects may occur. For example, the wording "such native sequence proteins can be made using standard recombinant and / or synthetic methods" indicates that native sequence proteins can be made using standard recombinant and synthetic methods or native sequence proteins can be made using standard recombinant methods or native sequence proteins can be made using synthetic methods.

[0116] As used herein, “treating” refers to a therapeutic use (i.e., on a subject having a given disease) and means reversing, alleviating, inhibiting the progress of one or more symptoms of such disorder or condition. Therefore, treatment does not only refer to a treatment that leads to a complete cure of the disease, but also to treatments that slow down the progression of the disease and / or prolong the survival of the subject.

[0117] As used herein, “preventing” means a prophylactic use (i.e., on a subject susceptible of developing a given disease and encompasses the treatment of relapsed AML patient.

[0118] As used herein, the terms “therapeutically effective amount” of the multifunctional binding protein or pharmaceutical composition thereof is meant a sufficient amount of the antibody-like multifunctional binding protein to treat said cancer disease, at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the polypeptides and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific polypeptide employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific polypeptide employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0119] As used herein, the term “subject” or “individual” or “patient” are used interchangeably and may encompass a human or a non-human mammal, rodent or non-rodent. The term includes, but is not limited to, mammals, e.g., humans including man, woman and child, other primates (monkey), pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats.

[0120] As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a pharmaceutically acceptable carrier” encompasses a plurality of pharmaceutically acceptable carriers, including mixtures thereof.

[0121] As used herein, “a plurality of’ may thus include « two » or « two or more ».

[0122] As used herein, “antibody” or “immunoglobulin” may refer to a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (X) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain generally includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). In particular, classes IgG, IgA, and IgD have three heavy chain constant region domains, which are designated CHI CH2, and CH3; and the IgM and IgE classes have four heavy chain constant region domains, CHI, CH2, CH3, and CH4. The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the antigen-binding fragment (Fab) of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain.

[0123] As used herein, when referring to “IgG” or “Immunoglobulin G” in general, IgGl, IgG2, IgG3 and IgG4 are included, unless defined otherwise. In particular, IgG is IgGl.

[0124] As used herein, the term “antibody-like” or “immunoglobulin-like” polypeptide may also refer to non-conventional or synthetic antigen-binding polypeptides or binding protein, including single domain antibodies and fragments thereof, in particular variable heavy chain of single domain antibodies, and chimeric, humanized, bispecific or multimeric antibodies.

[0125] As used herein, the term “multifunctional binding protein” encompass a multichain protein, including but not limited to antibody-like polypeptide or protein formats, which comprises at least one first variable region (e.g. a first immunoglobulin heavy chain variable domain (VH) and / or an immunoglobulin light chain variable domain (Vi.)) binding specifically to a human CD123 polypeptide, and at least one second variable region (e.g. a second immunoglobulin heavy chain variable domain (VH) and / or immunoglobulin light chain variable domain (VL)) binding specifically to a human NKp46 polypeptide. Although not limited specifically to a particular type of construct, one general embodiment is particularly considered throughout the specification: the polypeptide constructs reported in WO2015197593 and WO2017114694, each of which is incorporated herein by reference. In particular, the multifunctional binding protein such as those reported in WO2015197593 and WO2017114694, may encompass any construct comprising one or more polypeptide chains.

[0126] As used herein, the term “domain” may be any region of a protein, generally defined on the basis of sequence homologies or identities, which is related to a specific structural or functional entity. Accordingly, the term “region”, as used in the context of the present disclosure, is broader in that it may comprise additional regions beyond the corresponding domain.

[0127] As used herein, the terms “linker region”, “linker peptide” or “linker polypeptide” or “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 a Fc region to one or more variable regions, such as one or more antigen-binding domains. Although the term is not limited to a particular size or polypeptide length, such amino acid linkers are generally less than 50 amino acids in length, preferably less than 30 amino acids in length, for instance 20 or less than 20 amino acids in length, for instance 15 or less than 15 amino acids in length. Such amino acid linkers may optionally comprise all or part of an immunoglobulin polypeptide chain, such as all or part of a hinge region of an immunoglobulin. Alternatively, the amino acid linker may comprise a polypeptide sequence that is not derived from a hinge region of an immunoglobulin, or even that is not derived from an immunoglobulin heavy or light polypeptide chain.

[0128] As used herein, an immunoglobulin hinge region, or a fragment thereof, may thus be considered as a particular type of linker, which is derived from an immunoglobulin polypeptide chain.

[0129] As used herein, the term “hinge region” or “hinge” refers to a generally flexible region and born by the corresponding heavy chain polypeptides, and which separates the Fc and Fab portions of certain isotypes of immunoglobulins, more particularly of the IgG, IgA or IgD isotypes. Such hinge regions are known in the Art to depend upon the isotype of immunoglobulin which is considered. For native IgG, IgA and IgD isotypes, the hinge region thus separates the CHI domain and the CH2 domain and is generally cleaved upon papain digestion. On the other hand, the region corresponding to the hinge in IgM and IgE heavy chains is generally formed by an additional constant domain with lower flexibility. Additionally, the hinge region may comprise one or more cysteines involved in interchain disulfide bonds. The hinge region may also comprise one or more binding sites to a Fey receptor, in addition to FcyR binding sites bom by the CH2 domain, when applicable. Additionally, the hinge region may comprise one or more post-translational modification, such as one or more glycosylated residues depending on the isotype which is considered. Thus, it will be readily understood that the reference to the term “hinge” throughout the specification is not limited to a particular set of hinge sequences or to a specific location on the structure. Unless instructed otherwise, the hinge regions which are still particularly considered comprise all or part of a hinge from an immunoglobulin belonging to one isotype selected from: the IgG isotype, the IgA isotype and the IgD isotype; in particular the IgG isotype.

[0130] As used herein, the terms “CH domain”, or “CH domain”, or “constant domain”, can be used interchangeably and refer to any one or more heavy chain immunoglobulin constant domain(s). Such CH domains are natively folded as immunoglobulin-like domains, although they may be partly disordered in an isolated form (e.g., CHI domains when not associated with the constant domain of a light chain (CL)). Unless instructed otherwise, the term may thus refer to a CH 1 domain, a CH2 domain, a CH3 domain; or any combinations thereof.

[0131] As used herein, the terms “CHI domain”, or “CHI domain”, or “constant domain 1”, can be used interchangeably and refer to the corresponding heavy chain immunoglobulin constant domain 1.

[0132] As used herein, the term “CH2 domain”, or “CH2 domain”, or “constant domain 2” can be used interchangeably and refer to the corresponding heavy chain immunoglobulin constant domain 2.

[0133] As used herein, the term “CH3 domain”, or “CH3 domain”, or “constant domain 3” can be used interchangeably and refer to the corresponding heavy chain immunoglobulin constant domain 3.

[0134] As used herein, the term “CH2-CH3”, as in (CH2-CH3)A and (CH2-CH3)B, thus refers to a polypeptide sequence comprising an immunoglobulin heavy chain constant domain 2 (CH2) and an immunoglobulin heavy chain constant domain 3 (CH3).

[0135] As used herein, the term “CL domain”, or “CL domain” can be used interchangeably and refer to the corresponding light chain immunoglobulin constant domain. Unless instructed otherwise, this term may thus encompass a CL domain of the kappa (K or K) or lambda (X) class of immunoglobulin light chains, including all known subtypes (e.g. i, Xz, 3, and 7). In particular, when the CL domain is of the kappa class, it may also be referred herein as a CK or CK or Ck domain.

[0136] As used herein, the terms “pair C (CH1 / CL)”, or “paired C (CH1 / CL)” “refers to one constant heavy chain domain 1 and one constant light chain domain (e.g., a kappa (K or K) or lambda (X) class of immunoglobulin light chains) bound to one another by covalent or non- covalent bonds, preferably non-covalent bonds; thus forming a heterodimer. Unless specified otherwise, when the constant chain domains forming the pair are not present on a same polypeptide chain, this term may thus encompass all possible combinations. Preferably, the corresponding CHI and CL domains will thus be selected as complementary to each other, such that they form a stable pair C (CHI / CL).

[0137] Advantageously, when the binding protein comprises a plurality of paired C domains, such as one “pair Ci (CHI / CL)” and one “pair C2 (CHI / CL)”, each CHI and CL domain forming the pairs will be selected so that they are formed between complementary CHI and CL domains. Examples of complementary CHI and CL domains have been previously described in the international patent applications W02006064136 or WO2012089814 or WO2015197593A1.

[0138] Unless instructed otherwise, the terms “pair Ci (CH1 / CL)” or “pair C2 (CH1 / CL)” may refer to distinct constant pair domains (Ci and C2) formed by identical or distinct constant heavy 1 domains (CHI) and identical or distinct constant light chain domains (CL). Preferably, the terms “pair Ci (CH1 / CL)” or “pair C2 (Cnl / CL)” may refer to distinct constant pair domains (Ci and C2) formed by identical constant heavy 1 domains (CHI) and identical constant light chain domains (CL).

[0139] As used herein, the term “Fc region” or “fragment crystallizable region”, or alternatively “Fc portion”, encompasses all or parts of the “Fc domain”, which may thus include all or parts of an immunoglobulin hinge region (which natively bears a first binding site to FcyRs), a CH2 domain (which natively bears a second binding site to FcyRs), and a CH3 domain of an immunoglobulin (e.g. of an IgG, IgA or IgD immunoglobulin), and / or when applicable of a CH4 domain of an immunoglobulin (e.g. for IgM and IgE). Preferably, the Fc region includes all or parts of, at least, a CH2 domain and a CH3 domain, and optionally all or parts of an immunoglobulin hinge region. The term may thus refer to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region. The original immunoglobulin source of the native Fc is, in particular, of human origin and can be any of the immunoglobulins, although IgGl are preferred. Native Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 13 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgGl, IgG2, lgG3, IgGAl, and IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG. The term "native Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms. Under that terminology, a “Fc region” may thus comprise or consist of CH2-CH3 (e.g., (CH2-CH3)A or (CH2-CH3)B or a binding pair thereof, and optionally all or part of an immunoglobulin hinge region, comprising a binding site to a human FcyR. Unless specified otherwise, the term “Fc region” may refer to either a native or variant Fc region.

[0140] The term “Fc variant” as used herein refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interaction with the receptor, are known in the art. Thus, the term "Fc variant" can comprise a molecule or sequence that is humanized from a nonhuman native Fc. Furthermore, a native Fc comprises regions that can be removed because they provide structural features or biological activity that are not required for the antibody-like binding proteins of the invention. Thus, the term “Fc variant” comprises a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues has be modified, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0141] The fragment crystallizable (Fc) regions (e.g., native or variant) according to the present disclosure retain a capacity to bind to a human Fc-y receptor polypeptide (Fey) which generally occurs on native Fc regions through binding of the antibody Fc-hinge region. As a reference, overall structures of IgGl, IgG2, and IgG4 are similar with more than 90% sequence homology, the major differences residing in the hinge region and CH2 domain, which form primary binding sites to FcyRs. The hinge region also functions as a flexible linker between the Fab and Fc portion.

[0142] Fc regions having one or more amino acid modifications (e.g., substitutions, deletions, insertions) in one or more portions, which modifications increase the affinity and avidity of the variant Fc region for an FcyR (including activating and inhibitory FcyRs) are further considered as Fc regions. In some embodiments, said one or more amino acid modifications increase the affinity of the Fc region for FcyRIIIA and / or FcyRIIA. In another embodiment, the variant Fc region further specifically binds FcyRIIB with a lower affinity than does the Fc region of the reference parent antibody (e.g., an antibody having the same amino acid sequence as the antibody except for the one or more amino acid modifications in the Fc region). Hence, native and variant Fc regions considered herein generally comprise a domain (i.e., a CH2 domain) capable of binding to human CD16, e.g., a human Fc domain comprising N-linked glycosylation at amino acid residue N297 (according to EU numbering).

[0143] As used herein, the term “Fc-competent” thus refers to a binding protein that is capable of binding specifically to a FcyR, in particular of an activating FcyR, in particular to one selected from FcyRI (CD64a), FcyRIIa (CD32a), and FcyRIIIa (CD16a), and more particularly to FcyRIIIa (CD16a). Alternatively, several modifications are reported to directly affect the binding to FcyRs, including mutation on residues 297 (according to EU numbering), or alternatively on residues 234 and 235 in the lower hinge region (according to the EU numbering system).

[0144] As used herein, the term “Fc-silent” refers to a binding protein with a Fc region, wherein the Fc region lacks a binding site to a FcyR (e.g., a Fc region lacking a CH2 domain with said binding site and hinge region with said binding site); in particular FcyRI, FcyRIIa, and FcyRIIIa, and more particularly to FcyRIIIa (CD16a).

[0145] As used herein, the term “variable”, as in “variable domain”, refers to certain portions of the relevant binding protein which differ extensively in sequence between and among antibodies and are used in the specific recognition and binding of a particular antibody for its particular target. However, the variability is not evenly distributed throughout the entire variable domains of antibodies. The variability is concentrated in three segments called complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) also known as hypervariable regions, both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework (FR) regions or sequences.

[0146] As used herein, the term “VH domain”, or “VH domain” can be used interchangeably and refer to the corresponding heavy chain immunoglobulin variable domain.

[0147] As used herein, the term “VL domain”, or “VL domain” can be used interchangeably and refer to the corresponding light chain immunoglobulin variable domain.

[0148] When the VH or VL domains are associated to a first antigen-binding domain (ABD) or to a second antigen-binding domain, they may also be respectively referred herein as “Vnl” and “VL1”, or “VH2” and “VL2”.

[0149] The terms “binding pair V (VH / VL)”, “VH / VL pair” or “(VH / VL) pair” or “VL / VH pair” or “(VL / VH) pair” can be used interchangeably. Heavy chain and light chain variable domain can pair in parallel to form the antigen binding domains (ABDs). Each binding pair includes both a VH and a VL region. Unless instructed otherwise, these terms do not specify which immunoglobulin variable regions are VH or VL regions and which ABD will bind specifically the protein expressed on the surface of an immune effector cell or a target cell (e.g., NKp46 and CD123).

[0150] As used herein, the term “hypervariable region’ when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. This term may be substituted by the terms “Complementarity Determining Regions” or “CDRs”. Thus, as used herein “Complementarity Determining Regions” or “CDRs” refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR-L1, CDR-L2, CDR-L3 and CDR - Hl, CDR-H2, CDR-H3, respectively. A conventional antibody antigen-binding domain, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain variable region. Also, as used herein, “Framework Regions” (FRs) refer to amino acid sequences interposed between CDRs, i.e., to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR-L1, FR-L2, FR-L3, FR-L4, and FR-H1, FR-H2, FR-H3, FR-H4, respectively. Accordingly, the light chain variable domain may thus be designated as (FR-Ll)-(CDR-Ll)- (FR-L2)-(CDR-L2)-(FR-L3)-(CDR-L3)-(FR-L4) and the heavy chain variable domain may thus be designated as (FR-H1)-(CDR-H1)-(FR-H2)-(CDR-H2)-(FR-H3)-(CDR-H)-(FR4-H3).

[0151] The assignment of amino acids to each variable region domain is in accordance with the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991). Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chain variable regions or between different light chain variable regions are assigned the same number. CDRs 1, 2 and 3 of a VL domain are also referred to herein, respectively, as CDR-L1, CDR-L2 and CDR-L3. CDRs 1, 2 and 3 of a VH domain are also referred to herein, respectively, as CDR-H1, CDR- H2 and CDR-H3. If so noted, the assignment of CDRs can be in accordance with IMGT® (Lefranc et al., Developmental & Comparative Immunology 27:55-77; 2003) in lieu of Kabat. Numbering of the heavy chain constant region is via the EU index as set forth in Kabat (Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, 1987 and 1991). The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well- known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745. (“Contact” numbering scheme), Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(l):55-77 (“IMGT” numbering scheme), and Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (AHo numbering scheme, also known as the Honegger numbering scheme).

[0152] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[0153] As used herein, the CDRs of an antibody can be determined according to the numbering system called, “IMGT” described in Lefranc (1999), The Immunologist, vol. 7: 132-136 and Lefranc et al. (1999), Nucleic Acids Res., vol. 27:209-212.

[0154] As used herein, the CDRs of an antibody can be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops. Chothia and Lesk (1987), J. Mol. Biol., vol.196:901-917; Al-Lazikani et al. (1997), J. Mol. Biol., vol. 273:927-948; Chothia et al. (1992), J. Mol. Biol., vol. 227:799-817; Tramontane A et al. (1990), J. Mol. Biol. vol. 215(1): 175-82.

[0155] As used herein, the CDRs of an antibody can be determined according to the Honegger- Pluckthun numbering scheme described in Honnegger and Pluckthun (2001), J. Mol. Biol., vol. 309(3):657-670.

[0156] As used herein, the term “cytotoxicity” refers to the quality of a compound, such as the multifunctional binding protein and IL-2 polypeptide according to the present disclosure, to be toxic to tumoral cells. Cytotoxicity may be induced by different mechanisms of action and can thus be divided into cell-mediated cytotoxicity, apoptosis, antibody-dependent cell- mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC).

[0157] As used herein, the term “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a mechanism of cell-mediated immune defence whereby an effector cell of the immune system actively lyses a target cell, whose membrane-surface antigens have been bound by specific antibodies or the multifunctional binding protein of the present disclosure.

[0158] As used herein, the terms “proliferative disorders”, “hyper-proliferative disorders” and / or “cancer” not only refer to solid tumors, such as cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid and their distant metastases, but also include blood cancers, including tumors of the hematopoietic and lymphoid tissues, such as lymphomas, myelomas, and leukemias. Leukemias include, but are not limited to acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

[0159] As used herein, “Acute myelogenous leukemia (AML)” is a clonal disorder clinically presenting as increased proliferation of heterogeneous and undifferentiated myeloid blasts. Without wishing to be bound by the theory, the leukemic hierarchy is maintained by a small population of LSCs (Leukemic Stem Cells) (AML-LSCs), which have the distinct ability for self-renewal, and are able to differentiate into leukemic progenitors. These progenitors generate the large numbers of leukemic blasts readily detectable in patients at diagnosis and relapse, leading ultimately to mortality. AML-LSC have been commonly reported as quiescent cells, in contrast to rapidly dividing clonogenic progenitors.

[0160] Within the context of AML, the term “relapse” may in particular be defined as the reoccurrence of AML after complete remission. In that sense “complete remission” or “CR” may be defined as follows: normal values for neutrophil (>1.0*109 / L), haemoglobin level of lOg / dl and platelet count (>100*109 / L) and independence from red cell transfusion; blast cells less than 5%, no clusters or collections of blasts, and absence of Auer rods on bone marrow examination; and normal maturation of blood cells (morphology; myelogramme) and absence of extramedullary leukemia.

[0161] As used herein, “myelodysplastic syndromes” (“MDS”), formerly known as preleukemia, are a collection of hematological conditions that involve ineffective production (or dysplasia) of the myeloid class of blood cells. They represent a spectrum of clonal hematopoietic stem cell disorders characterized by progressive bone marrow failure and increased risk of progression to acute myeloid leukemia (“AML”, also known as “acute myelogenous leukemia”). The International Prognostic Scoring System (“IPSS”) is widely used to identify patients with high-risk features based on the severity of their cytopenias, bone marrow myeloblast percentage, and cytogenetic abnormalities. As used herein, a “pharmaceutically acceptable carrier” is intended to include any and all carrier (such as any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like) which is compatible with pharmaceutical administration, in particular parenteral administration. The use of such media and agents for pharmaceutically active substances are known. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the present disclosure. For example, preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are 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 buffered media. In a non-exhaustive manner, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0. IM (e.g., 0.05M) phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It should be stable under the conditions of manufacture and storage and will in an embodiment be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In certain embodiments, isotonic agents are included, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0162] As used herein, and unless instructed otherwise, the term “at least one” may encompass “one or more”, or even “two or more” (or “a plurality”). For instance, it may encompass 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,

[0163] 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,

[0164] 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,

[0165] 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 more than 100.

[0166] As used herein, and unless instructed otherwise, the term “less than...” may encompass all values from 0 to the corresponding threshold, For instance, it may encompass 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,

[0167] 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,

[0168] 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,

[0169] 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, when applicable.

[0170] By “isolated” nucleic acid molecule or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present disclosure. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. An isolated polynucleotide includes a polynucleotide molecule contained in cells that ordinarily contain the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present disclosure, as well as positive and negative strand forms, and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator. CD123 NKCE Binding Proteins

[0171] The binding protein of the combination therapy described herein (i.e., the CD123 NKCE / IL-2 combination) is characterized in that it comprises: (i) a first antigen-binding domain (ABD) comprising a variable region which binds specifically to a human CD123 polypeptide, (ii) a second antigen-binding domain (ABD) comprising a variable region which binds specifically to a human NKp46 polypeptide, and (iii) all or part of an immunoglobulin Fc region or variant thereof which binds to a human Fc-y receptor.

[0172] In some embodiments, the binding protein is characterized in that it comprises:

[0173] (i) a first antigen-binding domain (ABD) comprising a variable region which binds specifically to a human CD123 polypeptide and comprising at least one CDR selected from SEQ ID NO: 1 to SEQ ID NO: 12, or a variant thereof with one or more conservative substitution(s);

[0174] (ii) a second antigen-binding domain (ABD) comprising a variable region which binds specifically to a human NKp46 polypeptide, and (iii) all or part of an immunoglobulin Fc region or variant thereof which binds to a human Fc-y receptor polypeptide.

[0175] In some embodiments, the disclosure relates to a binding protein characterized in that it comprises:

[0176] (i) a first antigen-binding domain (ABD) comprising a variable region which binds specifically to a human CD 123 polypeptide,

[0177] (ii) a second antigen-binding domain (ABD) comprising a variable region which binds specifically to a human NKp46 polypeptide and comprising at least one CDR selected from SEQ ID NO: 13 to SEQ ID NO: 40, or a variant thereof with one or more conservative substitution(s); and

[0178] (iii) all or part of an immunoglobulin Fc region or variant thereof which binds to a human Fc-y receptor polypeptide.

[0179] In some embodiments, the binding protein is characterized in that it comprises:

[0180] (i) a first antigen-binding domain (ABD) comprising a variable region which binds specifically to a human CD123 polypeptide and comprising at least one CDR selected from SEQ ID NO: 1 to SEQ ID NO: 12, or a variant thereof with one or more conservative substitution(s); and

[0181] (ii) a second antigen-binding domain (ABD) comprising a variable region which binds specifically to a human NKp46 polypeptide and comprising at least one CDR selected from SEQ ID NO: 13 to SEQ ID NO: 40, or a variant thereof with one or more conservative substitution(s); and

[0182] (iii) all or part of an immunoglobulin Fc region or variant thereof which binds to a human Fc-y receptor polypeptide.

[0183] In some embodiments, the binding protein is characterized in that it comprises:

[0184] (i) a first antigen-binding domain (ABD) comprising a variable region which binds specifically to a human CD 123 polypeptide and comprising three CDRs selected from SEQ ID NO: 1 to SEQ ID NO: 12, or a variant thereof with one or more conservative substitution(s), and

[0185] (ii) a second antigen-binding domain (ABD) comprising a variable region which binds specifically to a human NKp46 polypeptide and comprising three CDRs selected from SEQ ID NO: 13 to SEQ ID NO: 40, or a variant thereof with one or more conservative substitution(s), and

[0186] (iii) all or part of an immunoglobulin Fc region or variant thereof which binds to a human Fc-y receptor polypeptide.

[0187] In some embodiments, the binding protein is characterized in that it comprises:

[0188] (i) a first antigen-binding domain (ABD) comprising a variable region which binds specifically to a human CD 123 polypeptide, and comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3 respectively), and

[0189] (ii) a second antigen-binding domain (ABD) comprising a variable region which binds specifically to a human NKp46 polypeptide, and comprising an immunoglobulin light chain variable region (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3 respectively), and

[0190] (iii) all or part of an immunoglobulin Fc region or variant thereof which binds to a human Fc-y receptor polypeptide.

[0191] In some embodiments, the binding protein is characterized in that it comprises:

[0192] (i) a first antigen-binding domain (ABD) comprising a variable region which binds specifically to a human CD 123 polypeptide, and comprising an immunoglobulin heavy chain variable region (VH) with three complementary determining regions, at least one being selected from SEQ ID NO: 1 to SEQ ID NO: 6, and an immunoglobulin light chain variable region (VL) with three complementary determining regions, at least one being selected from SEQ ID NO: 7 to SEQ ID NO: 12;

[0193] (ii) a second antigen-binding domain (ABD) comprising a variable region which binds specifically to a human NKp46 polypeptide, and comprising an immunoglobulin heavy chain variable region (VH) with three complementary determining regions at least one being selected from SEQ ID NO: 13 to SEQ ID NO: 26, and an immunoglobulin light chain variable region (VL) with three complementary determining regions at least one being selected from SEQ ID NO: 27 to SEQ ID NO: 40; and

[0194] (iii) all or part of an immunoglobulin Fc region or variant thereof which binds to a human Fc-y receptor polypeptide.

[0195] In some embodiments, the binding protein is characterized in that it comprises:

[0196] (i) a first antigen-binding domain (ABD) comprising a variable region which binds specifically to a human CD 123 polypeptide, and comprising an immunoglobulin heavy chain variable region (VH) with three complementary determining regions, at least two being selected from SEQ ID NO: 1 to SEQ ID NO: 6, and an immunoglobulin light chain variable region (VL) with three complementary determining regions at least two being selected from SEQ ID NO: 7 to SEQ ID NO: 12;

[0197] (ii) a second antigen-binding domain (ABD) comprising a variable region which binds specifically to a human NKp46 polypeptide, and comprising an immunoglobulin heavy chain variable region (VH) with three complementary determining regions at least two being selected from SEQ ID NO: 13 to SEQ ID NO: 26, and an immunoglobulin light chain variable region (VL) with three complementary determining regions at least two being selected from SEQ ID NO: 27 to SEQ ID NO: 40; and

[0198] (iii) all or part of an immunoglobulin Fc region or variant thereof which binds to a human Fc-y receptor polypeptide.

[0199] In some embodiments, the binding protein is characterized in that it comprises:

[0200] (i) a first antigen-binding domain (ABD) comprising a variable region which binds specifically to a human CD 123 polypeptide, and comprising an immunoglobulin heavy chain variable region (VH) with three complementary determining regions, selected from SEQ ID NO: 1 to SEQ ID NO: 6, and an immunoglobulin light chain variable region (VL) with three complementary determining regions selected from SEQ ID NO: 7 to SEQ ID NO: 12; (ii) a second antigen-binding domain (ABD) comprising a variable region which binds specifically to a human NKp46 polypeptide, and comprising an immunoglobulin heavy chain variable region (VH) with three complementary determining regions selected from SEQ ID NO: 13 to SEQ ID NO: 26, and an immunoglobulin light chain variable region (VL) with three complementary determining regions selected from SEQ ID NO: 27 to SEQ ID NO: 40; and

[0201] (iii) all or part of an immunoglobulin Fc region or variant thereof which binds to a human Fc-y receptor polypeptide.

[0202] In some embodiments, the binding protein is characterized in that the first ABD binds specifically to human CD 123 and comprises an immunoglobulin heavy chain variable domain (VH) comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 3 respectively or corresponding to the amino acid sequences of SEQ ID NO: 4 to SEQ ID NO: 6 respectively.

[0203] According to some particular embodiments of this first general object, the binding protein is characterized in that the first ABD binds specifically to human CD123 and comprises an immunoglobulin light chain variable domain (VL) comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 7 to SEQ ID NO: 9 respectively or corresponding to the amino acid sequences of SEQ ID NO: 10 to SEQ ID NO: 12 respectively.

[0204] According to some particular embodiments of this first general object, the binding protein is characterized in that the second ABD binds specifically to human NKp46 and comprises an immunoglobulin heavy chain variable domain (VH) comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 13 to SEQ ID NO: 15 respectively.

[0205] According to some particular embodiments of this first general object, the binding protein is characterized in that the second ABD binds specifically to human NKp46 and comprises an immunoglobulin heavy chain variable domain (VH) comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 16 to SEQ ID NO: 18 respectively.

[0206] According to some particular embodiments of this first general object, the binding protein is characterized in that the second ABD binds specifically to human NKp46 and comprises an immunoglobulin heavy chain variable domain (VH) comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 19 to SEQ ID NO: 21 respectively. According to some particular embodiments of this first general object, the binding protein is characterized in that the second ABD binds specifically to human NKp46 and comprises an immunoglobulin heavy chain variable domain (VH) comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 22 to SEQ ID NO: 24 respectively.

[0207] According to some particular embodiments of this first general object, the binding protein is characterized in that the second ABD binds specifically to human NKp46 and comprises an immunoglobulin heavy chain variable domain (VH) comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 25 and SEQ ID NO: 26 respectively.

[0208] According to some particular embodiments of this first general object, the binding protein is characterized in that the second ABD binds specifically to human NKp46 and comprises an immunoglobulin light chain variable domain (VL) comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 27 to SEQ ID NO: 29 respectively.

[0209] According to some particular embodiments of this first general object, the binding protein is characterized in that the second ABD binds specifically to human NKp46 and comprises an immunoglobulin light chain variable domain (VL) comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 30 to SEQ ID NO: 32 respectively.

[0210] According to some particular embodiments of this first general object, the binding protein is characterized in that the second ABD binds specifically to human NKp46 and comprises an immunoglobulin light chain variable domain (VL) comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 33 to SEQ ID NO: 35 respectively.

[0211] According to some particular embodiments of this first general object, the binding protein is characterized in that the second ABD binds specifically to human NKp46 and comprises an immunoglobulin light chain variable domain (VL) comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 36 to SEQ ID NO: 38 respectively.

[0212] According to some particular embodiments of this first general object, the binding protein is characterized in that the second ABD binds specifically to human NKp46 and comprises an immunoglobulin light chain variable domain (VL) comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 31 and SEQ ID NO: 40 respectively.

[0213] According to one embodiment, the disclosure relates to a binding protein comprising a first and a second antigen binding domains (ABDs) and all or part of an immunoglobulin Fc region or variant thereof, wherein each of said ABDs comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3); and wherein:

[0214] (i) the first ABD binds specifically to human CD123 and comprises:

[0215] - a VHI comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 3 respectively or corresponding to the amino acid sequences of SEQ ID NO: 4 to SEQ ID NO: 6 respectively, and

[0216] - a VLI comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 7 to SEQ ID NO: 9 respectively or corresponding to the amino acid sequences of SEQ ID NO: 10 to SEQ ID NO: 12 respectively;

[0217] (ii) the second ABD binds specifically to human NKp46 and comprises:

[0218] - a VH2 comprising a CDR-H1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 13 to SEQ ID NO: 15 respectively; the amino acid sequences of SEQ ID NO: 16 to SEQ ID NO: 18 respectively; the amino acid sequences of SEQ ID NO: 19 to SEQ ID NO: 21 respectively; the amino acid sequences of SEQ ID NO: 22 to SEQ ID NO: 24 respectively; or the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 25 and SEQ ID NO: 26 respectively; and

[0219] - a VL2 comprising a CDR-L1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 27 to SEQ ID NO: 29 respectively; the amino acid sequences of SEQ ID NO: 30 to SEQ ID NO: 32 respectively; the amino acid sequences of SEQ ID NO: 33 to SEQ ID NO: 35 respectively; the amino acid sequences of SEQ ID NO: 36 to SEQ ID NO: 38 respectively; or the amino acid sequences SEQ ID NO: 39, SEQ ID NO: 31 and SEQ ID NO: 40 respectively; and wherein all or part of the immunoglobulin Fc region or variant thereof binds to a human Fc-y receptor. According to another embodiment, the disclosure relates to a binding protein comprising a first and a second antigen binding domains (ABDs) and all or part of an immunoglobulin Fc region or variant thereof, wherein each of said ABDs comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3); and wherein:

[0220] (i) the first ABD comprises:

[0221] - a VH1 comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 88 to 90 respectively, and

[0222] - a VL1 comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 94 to 96 respectively; and

[0223] (ii) the second ABD comprises:

[0224] - a VH2 comprising a CDR-H1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 13 to 15 respectively; the amino acid sequences of SEQ ID NO: 16 to 18 respectively; the amino acid sequences of SEQ ID NO: 19 to 21 respectively; the amino acid sequences of SEQ ID NO: 22 to 24 respectively; or the amino acid sequences of SEQ ID NO: 16, 25 and 26 respectively; and

[0225] - a VL2 comprising a CDR-L1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 27 to 29 respectively; the amino acid sequences of SEQ ID NO: 30 to 32 respectively; the amino acid sequences of SEQ ID NO: 33 to 35 respectively; the amino acid sequences of SEQ ID NO: 36 to 38 respectively; or the amino acid sequences SEQ ID NO: 39, 31 and 40 respectively.

[0226] According to another embodiment, the disclosure relates to a binding protein comprising a first and a second antigen binding domains (ABDs) and all or part of an immunoglobulin Fc region or variant thereof, wherein each of said ABDs comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3); and wherein:

[0227] (i) the first ABD comprises: - a VH1 comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 91 to 93 respectively, and

[0228] - a VL1 comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 97 to 99 respectively; and

[0229] (ii) the second ABD comprises:

[0230] - a VH2 comprising a CDR-H1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 13 to 15 respectively; the amino acid sequences of SEQ ID NO: 16 to 18 respectively; the amino acid sequences of SEQ ID NO: 19 to 21 respectively; the amino acid sequences of SEQ ID NO: 22 to 24 respectively; or the amino acid sequences of SEQ ID NO: 16, 25 and 26 respectively; and

[0231] - a VL2 comprising a CDR-L1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 27 to 29 respectively; the amino acid sequences of SEQ ID NO: 30 to 32 respectively; the amino acid sequences of SEQ ID NO: 33 to 35 respectively; the amino acid sequences of SEQ ID NO: 36 to 38 respectively; or the amino acid sequences SEQ ID NO: 39, 31 and 40 respectively.

[0232] According to another embodiment, the disclosure relates to a binding protein comprising a first and a second antigen binding domains (ABDs) and all or part of an immunoglobulin Fc region or variant thereof, wherein each of said ABDs comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3); and wherein:

[0233] (i) the first ABD comprises:

[0234] - a VH1 comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 88 to 90 respectively, and

[0235] - a VL1 comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 94 to 96 respectively; and

[0236] (ii) the second ABD comprises:

[0237] - a VH2 comprising a CDR-H1, 2 and 3 corresponding to the amino acid sequences of SEQ ID NO: 103 to 105 respectively; and - a VL2 comprising a CDR-L1, 2 and 3 corresponding to the amino acid sequences of SEQ ID NO: 115 to 116 respectively.

[0238] According to another embodiment, the disclosure relates to a binding protein comprising a first and a second antigen binding domains (ABDs) and all or part of an immunoglobulin Fc region or variant thereof, wherein each of said ABDs comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3); and wherein:

[0239] (i) the first ABD comprises:

[0240] - a VH1 comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 91 to 93 respectively, and

[0241] - a VL1 comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 97 to 99 respectively; and

[0242] (ii) the second ABD comprises:

[0243] - a VH2 comprising a CDR-H1, 2 and 3 corresponding to the amino acid sequences of SEQ ID NO: 13 to 15 respectively; and

[0244] - a VL2 comprising a CDR-L1, 2 and 3 corresponding to the amino acid sequences of SEQ ID NO: 27 to 29 respectively.

[0245] It will be readily understood by the skilled in the Art that the above described binding protein may consist of one single polypeptide chain, or be a multimeric binding protein, and hence comprise a plurality (two or more) polypeptide chains.

[0246] According to some particular embodiments, the binding protein is a multimeric binding protein, and the two antigen-binding domains may be bom at least in part by distinct polypeptide chains.

[0247] Optionally, when the binding protein comprises a plurality of polypeptide chains, (e.g., two or three polypeptide chains), some of those polypeptide chains may be linked covalently. When two polypeptide chains are linked covalently, the covalent linker(s) may advantageously be selected from disulfide bridges, or any other covalent linker, including peptide bond(s) bridging one polypeptide chain with another, and / or linker peptide(s) bridging one polypeptide chain with another.

[0248] According to some particular embodiments, the binding protein is characterized in that it comprises three polypeptide chains (I), (II) and (III) that form two ABDs: VIA - CIA - L3 - (CH2-CH3)A (I)

[0249] VIB - CIB - L4 - (CH2-CH3)B- LI - V2A - C2A - 2 (II)

[0250] V2B - C2B (III) wherein :

[0251] VIA and VIB form a binding pair Vi (VHI / VLI);

[0252] V2A and V2B form a binding pair V2 (VH2 / VL2);

[0253] CIA and CIB form a pair Ci (CH1 / CL) and C2A and C2B form a pair C2 (CH1 / CL) wherein CHI is an immunoglobulin heavy chain constant domain 1 and CL is an immunoglobulin light chain constant domain;

[0254] (Cu2-CH3)Aand (CH2-CH3)B are identical or different, and comprise an immunoglobulin heavy chain constant domain 2 (CH2) and an immunoglobulin heavy chain constant domain 3 (CH3);

[0255] LI, L2, L3, L4 are optional independent amino acid linkers, which may be identical or different.

[0256] In some embodiments, the (CH2-CH3)A and (CH2-CH3)B comprise each at least one identical CH2 domain, such as a CH2 domain corresponding to the amino acid sequence of SEQ ID NO: 71.

[0257] In some embodiments, the (Cu2-CH3)Aand (CH2-CH3)B are identical or different and may comprise a polypeptide sequence selected from amino acid sequences of SEQ ID NO: 69 or SEQ ID NO: 70.

[0258] In some embodiments, some of Li, L2, L3 and L4 may be identical or different, and may comprise all or part of an amino acid sequence selected from SEQ ID NO: 74 to SEQ ID NO: 79; for example, one or more than four consecutive amino acids of an amino acid sequence selected from SEQ ID NO: 74 to SEQ ID NO: 79.

[0259] According to some particular embodiments, some of Li, L2, L3 and L4 may be identical or different, and may comprise all or part of an immunoglobulin hinge region, such as one selected from amino acid sequences SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78 and / or SEQ ID NO: 79; for example four or more than four consecutive amino acids of an immunoglobulin hinge region, such as one selected from amino acid sequences SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78 and / or SEQ ID NO: 79.

[0260] According to some more particular embodiments, L2, L3 and L4 may be identical or different, and may comprise all or part of an immunoglobulin hinge region, such as one selected from sequences SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78 and / or SEQ ID NO: 79; for example, four or more than four consecutive amino acids of an immunoglobulin hinge region, such as one selected from sequences SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78 and / or SEQ ID NO: 79.

[0261] According to some more particular embodiments, L2, L3 and L4 may be identical or different, and may comprise all or part of an immunoglobulin hinge region, such as one selected from sequences SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78 and / or SEQ ID NO: 79 (for example four or more than four consecutive amino acids of an immunoglobulin hinge region, such as one selected from sequences SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78 and / or SEQ ID NO: 79), and Li may comprise all or part of the linker which corresponds to the amino acid sequence of SEQ ID NO: 76.

[0262] According to some particular embodiments, the binding protein is characterized in that it comprises three polypeptide chains (I), (II) and (III) that form two ABDs, as defined below:

[0263] VIA - CIA - L3 - (CH2-CH3)A (I)

[0264] VIB - CIB - L4 - (CH2-CH3)B - LI - V2A - C2A - L2 (II)

[0265] V2B - C2B (III) wherein:

[0266] VIA and VIB form a binding pair Vi (VHI / VLI), which binds specifically to a human CD 123 polypeptide;

[0267] V2A and V2B form a binding pair V2 (VH2 / VL2), which binds specifically to a human NKp46 polypeptide;

[0268] CIA and CIB form a pair Ci (CH1 / CL) and C2A and C2B form a pair C2 (CH1 / CL) wherein CH I is an immunoglobulin heavy chain constant domain 1 and CL is an immunoglobulin light chain constant domain;

[0269] (Cu2-CH3)Aand (CH2-CH3)B are identical or different, and comprise an immunoglobulin heavy chain constant domain 2 (CH2) and an immunoglobulin heavy chain constant domain 3 (CH3);

[0270] LI, L2, L3, L4 are optional independent amino acid linkers, which may be identical or different.

[0271] In one embodiment, the binding protein is characterized in that it comprises three polypeptide chains (I), (II) and (III) that form two ABDs, as defined below:

[0272] VIA - CIA - Hingei - (CH2-CH3)A (I)

[0273] VIB - CIB - Hinge2 - (CH2-CH3)B - LI - V2A - C2A - Hinges (II) V2B - C2B QU) wherein :

[0274] VIA and VIB form a binding pair Vi (VHI / VLI);

[0275] V2A and V2B form a binding pair V2 (VH2 / VL2);

[0276] CIA and CIB form a pair Ci (CH1 / CL) and C2A and C2B form a pair C2 (CHI / CL) wherein CHI is an immunoglobulin heavy chain constant domain 1 and CL is an immunoglobulin light chain constant domain;

[0277] Hingei, Hinge2 and Hinges are identical or different and correspond to all or part of an immunoglobulin hinge region;

[0278] (Cu2-CH3)Aand (CH2-CH3)B are identical or different, and comprise an immunoglobulin heavy chain constant domain 2 (CH2) and an immunoglobulin heavy chain constant domain 3 (CH3);

[0279] LI is an amino acid linker.

[0280] In one embodiment, the binding protein is characterized in that it comprises three polypeptide chains (I), (II) and (III) that form two ABDs, as defined below:

[0281] VIA - CIA - Hingei - (CH2-CH3)A (I)

[0282] VIB - CIB - Hinge2 - (CH2-CH3)B - LI - V2A - C2A - Hinges (II)

[0283] V2B - C2B (III) wherein :

[0284] VIA and VIB form a binding pair Vi (VHI / VLI) which binds specifically to a human CD 123 polypeptide;

[0285] V2A and V2B form a binding pair V2 (VH2 / VL2) which binds specifically to a human NKp46 polypeptide;

[0286] CIA and CIB form a pair Ci (CH1 / CL) and C2A and C2B form a pair C2 (CHI / CL) wherein CHI is an immunoglobulin heavy chain constant domain 1 and CL is an immunoglobulin light chain constant domain;

[0287] Hingei, Hinge2 and Hinges are identical or different and correspond to all or part of an immunoglobulin hinge region;

[0288] (Cu2-Cu3)Aand (CH2-CH3)B are identical or different, and comprise an immunoglobulin heavy chain constant domain 2 (CH2) and an immunoglobulin heavy chain constant domain 3

[0289] (CH3);

[0290] LI is an amino acid linker. In some embodiments of the binding protein, the polypeptide chains (I), (II) and (III) are characterized in that:

[0291] CIA comprises a CL domain;

[0292] CIB comprises a CHI domain;

[0293] C2A comprises a CHI domain;

[0294] C2B comprises a CL domain.

[0295] In some embodiments of the binding protein, the polypeptide chains (I), (II) and (III) are characterized in that:

[0296] CIA comprises a CHI domain;

[0297] CIB comprises a CL domain;

[0298] C2A comprises a CL domain;

[0299] C2B comprises a CHI domain.

[0300] According to some of those particular embodiments of the binding protein, the polypeptide chains (I), (II) and (III) are characterized in that:

[0301] CIA comprises a CHI domain;

[0302] CIB comprises a CL domain;

[0303] C2A comprises a CHI domain;

[0304] C2B comprises a CL domain.

[0305] According to some of those particular embodiments of the binding protein, the polypeptide chains (I), (II) and (III) are characterized in that:

[0306] CIA comprises a CL domain;

[0307] CIB comprises a CHI domain;

[0308] C2A comprises a CL domain;

[0309] C2B comprises a CHI domain.

[0310] In some embodiments, CL and CHI domains which form CIA, CIB, C2A, and C2B may be identical or different. Hence in some embodiments of the binding protein, the polypeptide chains (I), (II) and (III) are characterized in that:

[0311] CIA and C2A are identical and comprise a CL domain; or

[0312] CIA and C2B are identical and comprise a CL domain; or

[0313] CIB and C2A are identical and comprise a CL domain; or

[0314] CIB and C2B are identical and comprise a CL domain. In some embodiments of the binding protein, the polypeptide chains (I), (II) and (III) are characterized in that:

[0315] CIA and C2A are identical and comprise a CHI domain; or

[0316] CIA and C2B are identical and comprise a CHI domain; or

[0317] CIB and C2A are identical and comprise a CHI domain; or

[0318] CIB and C2B are identical and comprise a CHI domain.

[0319] In some embodiments of polypeptide chains (I), (II) and (III): VIA is a VH and VIB is a VL.

[0320] In some embodiments of polypeptide chains (I), (II) and (III): VIA is a VL and VIB is a VH.

[0321] In some embodiments of polypeptide chains (I), (II) and (III): V2A is a VH and V2B is a VL.

[0322] In some embodiments of polypeptide chains (I), (II) and (III): V2A is a VL and V2B is a VH.

[0323] In some embodiments of polypeptide chains (I), (II) and (III): VIA is a VH and VIB is a VL; and V2A is a VH and V2B is a VL.

[0324] In some embodiments of polypeptide chains (I), (II) and (III): VIA is a VL and VIB is a VH; and V2A is a VH and V2B is a VL.

[0325] In some embodiments of polypeptide chains (I), (II) and (III): VIA is a VH and VIB is a VL; and V2A is a VL and V2B is a VH.

[0326] In some embodiments of polypeptide chains (I), (II) and (III): VIA is a VL and VIB is a VH; and V2A is a VL and V2B is a VH.

[0327] In some embodiment of the binding protein, VIA is VLI and VIB is VHI; and V2A is VH2 and V2B is VL2.

[0328] In some embodiment of the binding protein, VIA is VLI and VIB is VHL

[0329] In some embodiment of the binding protein, V2A is VH2 and V2B is VL2.

[0330] In some embodiment of the binding protein, the polypeptide chains (I), (II) and (III) are characterized in that:

[0331] CIB is an immunoglobulin heavy chain constant domain 1 (CHI);

[0332] C2A is an immunoglobulin heavy chain constant domain 1 (CHI);

[0333] CL corresponds to an immunoglobulin kappa light chain constant domain (CK);

[0334] (CH2-CH3)A corresponds to the amino acid sequence of SEQ ID NO: 69;

[0335] (CH2-CH3)B corresponds to the amino acid sequence of SEQ ID NO: 70; L2 or Hingei corresponds to the amino acid sequence of SEQ ID NO: 74;

[0336] L3 or Hinge? corresponds to the amino acid sequence of SEQ ID NO: 75;

[0337] L4 or Hinges corresponds to the amino acid sequence of SEQ ID NO: 77.

[0338] Li corresponds to the amino acid sequence of SEQ ID NO: 76.

[0339] In some embodiment of the binding protein, the polypeptide chains (I), (II) and (III) are characterized in that:

[0340] CIB is an immunoglobulin heavy chain constant domain 1 (CHI);

[0341] C2A is an immunoglobulin heavy chain constant domain 1 (CHI);

[0342] CL corresponds to an immunoglobulin kappa light chain constant domain (CK);

[0343] (CH2-CH3)A corresponds to the amino acid sequence of SEQ ID NO: 69 ;

[0344] (CH2-CH3)B corresponds to the amino acid sequence of SEQ ID NO: 70;

[0345] Hingei corresponds to the amino acid sequence of SEQ ID NO: 74;

[0346] Hinge2 corresponds to the amino acid sequence of SEQ ID NO: 75;

[0347] Hinges corresponds to the amino acid sequence of SEQ ID NO: 77;

[0348] Li corresponds to the amino acid sequence of SEQ ID NO: 76.

[0349] In some embodiment of the binding protein:

[0350] (a) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO:9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 27; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 28; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29;

[0351] (b) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32;

[0352] (c) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 34; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35;

[0353] (d) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 37; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38;

[0354] (e) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 39; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40; (f) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 13; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 27; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 28; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29;

[0355] (g) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32;

[0356] (h) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 19; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35;

[0357] (i) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 22; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38;

[0358] (j) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 39; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0359] In some embodiments of the binding protein:

[0360] (a) VHI and VLI corresponds to the amino acid sequences of SEQ ID NO: 41 and 43 respectively or corresponds to the amino acid sequences of SEQ ID NO: 42 and 44 respectively; and / or

[0361] (b) VH2 and VL2 corresponds 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; the amino acid sequences of SEQ ID NO: 51 and 59 respectively; or the amino acid sequences of SEQ ID NO: 52 and 60 respectively.

[0362] In some embodiments of the binding protein: (a) VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 45; VL2 comprises the amino acid sequence of SEQ ID NO: 53;

[0363] (b)Vm comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 46; VL2 comprises the amino acid sequence of SEQ ID NO: 54;

[0364] (C)VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 47; VL2 comprises the amino acid sequence of SEQ ID NO: 55;

[0365] (d)Vm comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 48; VL2 comprises the amino acid sequence of SEQ ID NO: 56;

[0366] (e)Vm comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 49; VL2 comprises the amino acid sequence of SEQ ID NO: 57;

[0367] (f) VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 50; VL2 comprises the amino acid sequence of SEQ ID NO: 58;

[0368] (g)Vm comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 51; VL2 comprises the amino acid sequence of SEQ ID NO: 59;

[0369] (h)Vm comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 52; VL2 comprises the amino acid sequence of SEQ ID NO: 60;

[0370] (i) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 45; VL2 comprises the amino acid sequence of SEQ ID NO: 53;

[0371] (j) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 46; VL2 comprises the amino acid sequence of SEQ ID NO: 54;

[0372] (k) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 47; VL2 comprises the amino acid sequence of SEQ ID NO: 55; (l) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 48; VL2 comprises the amino acid sequence of SEQ ID NO: 56;

[0373] (m) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 49; VL2 comprises the amino acid sequence of SEQ ID NO: 57;

[0374] (n) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 50; VL2 comprises the amino acid sequence of SEQ ID NO: 58;

[0375] (o) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 51; VL2 comprises the amino acid sequence of SEQ ID NO: 59;

[0376] (p) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 52; VL2 comprises the amino acid sequence of SEQ ID NO: 60.

[0377] In some embodiments, the binding protein comprises at least two polypeptide chains linked by at least one disulfide bridge.

[0378] In some embodiments of the binding protein, the polypeptide chains (I), (II) and (III) are characterized in that: polypeptide chain (I) is covalently linked to polypeptide chain (II), in particular covalently liked to polypeptide (II) by one or more disulfide bonds.

[0379] According to some of those particular embodiments of the binding protein, the polypeptide chains (I), (II) and (III) are characterized in that: polypeptide chain (II) is covalently linked to polypeptide chain (III), by one or more disulfide bonds.

[0380] In some embodiments, the polypeptide chains (I) and (II) are linked by at least one disulfide bridge between CIA and Hinge2 and / or wherein the polypeptide chains (II) and (III) are linked by at least one disulfide bridge between Hinges and C2B.

[0381] In some embodiments, the binding protein is characterized in that the Fc region or variant thereof (e.g. (CH2-CH3)A or (CH2-CH3)B or Hingei - (CH2-CH3)A or Hinge2 - (CH2- CH3)B) which binds to a human Fc-y receptor polypeptide, comprises a CH2 heavy chain constant domain with a N-linked glycosylation at residue N297 according to EU numbering. In some embodiments, the binding protein is characterized in that the residue N297 of the Fc region or variant thereof according to EU numbering comprises a N-linked glycosylation.

[0382] In some embodiments, the binding protein is characterized in that all or part of the Fc region or variant thereof binds to a human Fc-y receptor polypeptide. In some embodiments, the binding protein is characterized in that all or part of the Fc region or variant thereof binds to a human CD16A (FcyRIII) polypeptide.

[0383] In one embodiment, the binding protein comprises: a polypeptide comprising an amino acid sequence of SEQ ID NO: 61, a polypeptide comprising an amino acid sequence of SEQ ID NO: 62 and a polypeptide comprising an amino acid sequence of SEQ ID NO: 63, or a variant thereof with at least 80% of sequence identity; or a polypeptide comprising an amino acid sequence of SEQ ID NO: 64, a polypeptide comprising an amino acid sequence of SEQ ID NO: 65 and a polypeptide comprising an amino acid sequence of SEQ ID NO: 66 or a variant thereof with at least 80% of sequence identity; and / or a polypeptide comprising an amino acid sequence of SEQ ID NO: 61 or 64, a polypeptide comprising an amino acid sequence of SEQ ID NO: 62 or 65 and a polypeptide comprising an amino acid sequence of SEQ ID NO: 63 or 66, or a variant thereof with at least 80% of sequence identity.

[0384] In some embodiments, the binding protein comprises: a polypeptide comprising an amino acid sequence SEQ ID NO: 61, a polypeptide comprising an amino acid sequence SEQ ID NO: 62 and a polypeptide comprising an amino acid sequence SEQ ID NO: 63 or a variant thereof with at least 80% of sequence identity; or a polypeptide comprising an amino acid sequence of SEQ ID NO: 64, a polypeptide comprising an amino acid sequence of SEQ ID NO: 65 and a polypeptide comprising an amino acid sequence of SEQ ID NO: 66 or a variant thereof with at least 80% of sequence identity. In some embodiments, the binding protein comprises: a polypeptide comprising an amino acid sequence of SEQ ID NO: 61, a polypeptide comprising a sequence SEQ ID NO: 62 and a polypeptide comprising an amino acid sequence of SEQ ID NO: 63 or a variant thereof with at least 80% of sequence identity.

[0385] In some embodiments, the binding protein comprises: a polypeptide comprising an amino acid sequence of SEQ ID NO: 64, a polypeptide comprising an amino acid sequence of SEQ ID NO: 65 and a polypeptide comprising an amino acid sequence of SEQ ID NO: 66 or a variant thereof with at least 80% of sequence identity.

[0386] In some embodiments, the binding protein comprises: a polypeptide comprising an amino acid sequence of SEQ ID NO: 61, a polypeptide comprising an amino acid sequence of SEQ ID NO: 62 and a polypeptide comprising an amino acid sequence of SEQ ID NO: 63 or a variant thereof with at least 90% of sequence identity.

[0387] In some embodiments, the binding protein comprises: a polypeptide comprising an amino acid sequence of SEQ ID NO: 64, a polypeptide comprising a amino acid sequence of SEQ ID NO: 65 and a polypeptide comprising an amino acid sequence of SEQ ID NO: 66 or a variant thereof with at least 90% of sequence identity.

[0388] In some embodiments, the binding protein comprises: a polypeptide comprising an amino acid sequence of SEQ ID NO: 61, a polypeptide comprising an amino acid sequence of SEQ ID NO: 62 and a polypeptide comprising an amino acid sequence of SEQ ID NO: 63 or a variant thereof with at least 95% of sequence identity.

[0389] In some embodiments, the binding protein comprises: a polypeptide comprising an amino acid sequence of SEQ ID NO: 64, a polypeptide comprising a amino acid sequence of SEQ ID NO: 65 and a polypeptide comprising an amino acid sequence of SEQ ID NO: 66 or a variant thereof with at least 95% of sequence identity.

[0390] In some embodiments, the binding protein comprises: a polypeptide (I) comprising an amino acid sequence of SEQ ID NO: 61, a polypeptide (II) comprising an amino acid sequence of SEQ ID NO: 62 and a polypeptide (III) comprising an amino acid sequence of SEQ ID NO: 63.

[0391] In some embodiments, the binding protein comprises:

[0392] - polypeptide (I) consisting of an amino acid sequence of SEQ ID NO: 61;

[0393] - polypeptide (II) consisting of an amino acid sequence of SEQ ID NO: 62; and

[0394] - polypeptide (III) consisting of an amino acid sequence of SEQ ID NO: 63. In some embodiments, the binding protein comprises: a polypeptide (I) comprising an amino acid sequence of SEQ ID NO: 64, a polypeptide (II) comprising an amino acid sequence of SEQ ID NO: 65 and a polypeptide (III) comprising an amino acid sequence of SEQ ID NO: 66.

[0395] In some embodiments, the binding protein comprises:

[0396] - polypeptide (I) consisting of an amino acid sequence of SEQ ID NO: 64;

[0397] - polypeptide (II) consisting of an amino acid sequence of SEQ ID NO: 65; and

[0398] - polypeptide (III) consisting of an amino acid sequence of SEQ ID NO: 66.

[0399] In some variants of those embodiments, the binding protein comprises polypeptide sequences derived from immunoglobulins chains (in particular immunoglobulins of the IgG type), and / or amino acid sequences selected from any one of SEQ ID NO: 1 to SEQ ID NO: 79, which may thus include any variant sequence with conservative substitutions, and / or any variant with a degree of percent sequence identity with a reference sequence; especially a reference sequence derived from an immunoglobulin chain.

[0400] In some embodiments, the binding protein comprises polypeptide sequences derived from immunoglobulins chains of the IgG type, in particular of the IgGl, IgG2, IgG3 or IgG4 type, preferably of the IgGl type.

[0401] When variants of Fc and constant regions and non-CDRs polypeptide sequences from a variable region are considered herein, they may consist of Fc and constant regions and non-CDRs polypeptide sequences having at least 80% of sequence identity with a reference polypeptide sequence; more particularly having at least 90% of sequence identity with a reference polypeptide sequence; and preferably having at least 95% of sequence identity with a reference polypeptide sequence.

[0402] Alternatively, when variants of polypeptide sequences include CDR polypeptide sequences (e.g., CDR1, CDR2, and CDR3 from either one of a VH or VL domain), it will be understood herein that those variants do not have modifications on their CDR polypeptide sequences.

[0403] In some embodiments, the binding protein comprises an amino acid sequence having at least 80% of sequence identity with an amino acid sequence selected from SEQ ID NO: 67 to 73. In some embodiments, the binding protein comprises a amino acid sequence having at least 90% of sequence identity with an amino acid sequence selected from SEQ ID NO: 67 to 73.

[0404] In some embodiments, the binding protein comprises an amino acid sequence having at least 95% of sequence identity with an amino acid sequence selected from SEQ ID NO: 67 to 73.

[0405] In some embodiments, the binding protein comprises a Fc region or variant thereof having at least 80% of sequence identity with an amino acid sequence selected from SEQ ID NO: 69 to 73.

[0406] In some embodiments, the binding protein comprises a Fc region or variant thereof having at least 90% of sequence identity with an amino acid sequence selected from SEQ ID NO: 69 to 73.

[0407] In some embodiments, the binding protein comprises a Fc region or variant thereof having at least 95% of sequence identity with an amino acid s sequence selected from SEQ ID NO: 69 to 73.

[0408] In some embodiments, the binding protein comprises a Fc region or variant thereof with a CH2-CH3 domain having at least 80% of sequence identity with an amino acid sequence selected from SEQ ID NO: 69 or 70; or alternatively comprises a Fc region or variant thereof with a CH2 domain having at least 80% of sequence identity with an amino acid sequence of SEQ ID NO: 71; or alternatively comprises a Fc region or variant thereof with a CH3 domain having at least 80% of sequence identity with an amino acid sequence of SEQ ID NO: 72 or 73.

[0409] In some embodiments, the binding protein comprises a Fc region or variant thereof with a CH2-CH3 domain having at least 90% of sequence identity with an amino acid sequence selected from SEQ ID NO: 69 or 70; or alternatively comprises a Fc region or variant thereof with a CH2 domain having at least 90% of sequence identity with an amino acid sequence SEQ ID NO: 71; or alternatively comprises a Fc region or variant thereof with a CH3 domain having at least 90% of sequence identity with an amino acid sequence of SEQ ID NO: 72 or 73.

[0410] In some embodiments, the binding protein comprises a Fc region or variant thereof with a CH2-CH3 domain having at least 95% of sequence identity with an amino acid sequence selected from SEQ ID NO: 69 or 70; or alternatively comprises a Fc region or variant thereof with a CH2 domain having at least 95% of sequence identity with an amino acid sequence of SEQ ID NO: 71; or alternatively comprises a Fc region or variant thereof with a CH3 domain having at least 95% of sequence identity with an amino acid sequence of SEQ ID NO: 72 or 73.

[0411] Preferably, the multispecific binding proteins of the present disclosure are bispecific binding proteins.

[0412] The disclosure further relates to a pharmaceutical composition comprising a binding protein as defined above, and a pharmaceutically acceptable carrier.

[0413] Hence, in one embodiment, the disclosure relates to a pharmaceutical composition comprising a binding protein, and a pharmaceutically acceptable carrier, said binding protein comprising:

[0414] (i) a first antigen-binding domain (ABD) comprising a variable region which binds specifically to a human CD 123 polypeptide, (ii) a second antigen-binding domain (ABD) comprising a variable region which binds specifically to a human NKp46 polypeptide, and (iii) all or part of an immunoglobulin fragment crystallizable (Fc) region or variant thereof which binds to a human Fc-y receptor polypeptide.

[0415] Hence, in one embodiment, the disclosure relates to a pharmaceutical composition comprising the binding protein defined above, and a pharmaceutically acceptable carrier, said binding protein comprising a first and a second antigen binding domains (ABDs) and all or part of an immunoglobulin Fc region or variant thereof, wherein each of said ABDs comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3); and wherein:

[0416] (i) the first ABD binds specifically to human CD123 and comprises:

[0417] - a VHI comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 1 to 3 respectively or corresponding to the amino acid sequences of SEQ ID NO: 4 to 6 respectively, and

[0418] - a VLI comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 7 to 9 respectively or corresponding to the amino acid sequences of SEQ ID NO: 10 to 12 respectively;

[0419] (ii) the second ABD binds specifically to human NKp46 and comprises: - a VH2 comprising a CDR-H1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 13 to 15 respectively; the amino acid sequences of SEQ ID NO: 16 to 18 respectively; the amino acid sequences of SEQ ID NO: 19 to 21 respectively; the amino acid sequences of SEQ ID NO:22 to 24 respectively; or the amino acid sequences of SEQ ID NO: 16, 25 and 26 respectively; and

[0420] - a VL2 comprising a CDR-L1, 2 and 3 corresponding to: the amino acid sequences of of SEQ ID NO:27 to 29 respectively; the amino acid sequences of SEQ ID NO:30 to 32 respectively; the amino acid sequences of SEQ ID NO:33 to 35 respectively; the amino acid sequences of SEQ ID NO:36 to 38 respectively; or the amino acid sequences of SEQ ID NO:39, 31 and 40 respectively; and wherein all or part of the immunoglobulin Fc region or variant thereof to a human Fc-y receptor

[0421] Interleukin 2 Polypeptides and Variants and Conjugates Thereof

[0422] Interleukin 2 (IL-2) is a pleiotropic type-1 cytokine whose structure comprises a 15.5 kDa four a-helix bundle. The precursor form of IL-2 is 153 amino acid residues in length, with the first 20 amino acids forming a signal peptide and residues 21-153 forming the mature form. IL-2 is produced primarily by CD4+ T cells post antigen stimulation and to a lesser extent, by CD8+ cells, Natural Killer (NK) cells, and Natural killer T (NKT) cells, activated dendritic cells (DCs), and mast cells. IL-2 signaling occurs through interaction with specific combinations of IL-2 receptor (IL-2R) subunits, IL-2Ra (also known as CD25), IL-2RP (also known as CD122), and IL-2Ry (also known as CD132). Interaction of IL-2 with the IL-2Ra forms the “low-affinity” IL-2 receptor complex with a Kd of about 10'8M. Interaction of IL-2 with IL-2RP and IL-2R.y forms the “intermediate-affinity” IL-2 receptor complex with a Kd of about 10'9M. Interaction of IL-2 with all three subunits, IL-2Ra, IL-2RP, and IL-2Ry, forms the “high-affinity” IL-2 receptor complex with a Kd of about 10'11M.

[0423] In some instances, IL-2 signaling via the “high-affinity” IL-2RaPy complex modulates the activation and proliferation of regulatory T cells. Regulatory T cells, or CD4+CD25+Foxp3+ regulatory T (Treg) cells, mediate maintenance of immune homeostasis by suppression of effector cells such as CD4+ T cells, CD8+ T cells, B cells, NK cells, and NKT cells. In some instances, Treg cells are generated from the thymus (tTreg cells) or are induced from naive T cells in the periphery (pTreg cells). In some cases, Treg cells are considered as the mediator of peripheral tolerance. Indeed, in one study, transfer of CD25- depleted peripheral CD4+ T cells produced a variety of autoimmune diseases in nude mice, whereas cotransfer of CD4+CD25+ T cells suppressed the development of autoimmunity (Sakaguchi, et al., “Immunologic self-tolerance maintained by activated T cells expressing IL- 2 receptor alpha-chains (CD25),” J. Immunol. 155(3): 1151-1164 (1995)). Augmentation of the Treg cell population down-regulates effector T cell proliferation and suppresses autoimmunity and T cell anti-tumor responses.

[0424] IL-2 signaling via the “intermediate-affinity” IL-2R.Py complex modulates the activation and proliferation of CD8+ effector T (Teff) cells, NK cells, and NKT cells. CD8+ Teff cells (also known as cytotoxic T cells, Tc cells, cytotoxic T lymphocytes, CTLs, T-killer cells, cytolytic T cells, Tcon, or killer T cells) are T lymphocytes that recognize and kill damaged cells, cancerous cells, and pathogen-infected cells. NK and NKT cells are types of lymphocytes that, similar to CD8+ Teff cells, target cancerous cells and pathogen-infected cells.

[0425] Accordingly, described herein are IL-2 polypeptides and conjugates thereof, for use in combination with the binding proteins described herein (i.e., the anti-CD123 x NKp46 binding proteins, i.e., the CD123 NKCE).

[0426] In some embodiments, the IL-2 polypeptide is a wild-type IL-2 polypeptide. In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 84. In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 84.

[0427] In some embodiments, the IL-2 polypeptide is a wild-type IL-2 polypeptide. In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 85. In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 85.

[0428] IL-2 Variants and Conjugates

[0429] In some embodiments, the IL-2 polypeptide is a truncated variant. In some embodiments, the truncation is an N-terminal deletion. In other embodiments, the truncation is a C-terminal deletion. In additional embodiments, the truncation comprises both N-terminal and C-terminal deletions. For example, the truncation can be a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues from either the N-terminus or the C-terminus, or both termini of an IL-2 polypeptide described herein. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 2 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 3 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 4 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 5 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 6 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 7 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 8 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 9 residues. In some cases, the IL-2 polypeptide comprises an N- terminal deletion of at least or about 10 residues.

[0430] In some embodiments, the IL-2 polypeptide is a functionally active fragment. In some cases, the functionally active fragment comprises IL-2 region 10-133, 20-133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, wherein the residue positions are in reference to the positions in SEQ ID NO: 84. In some cases, the functionally active fragment comprises IL-2 region 10-133, wherein the residue positions are in reference to the positions in SEQ ID NO: 84. In some cases, the functionally active fragment comprises IL-2 region 20- 133, wherein the residue positions are in reference to the positions in SEQ ID NO: 84. In some cases, the functionally active fragment comprises IL-2 region 30-133, wherein the residue positions are in reference to the positions in SEQ ID NO: 84. In some cases, the functionally active fragment comprises IL-2 region 10-125, wherein the residue positions are in reference to the positions in SEQ ID NO: 84. In some cases, the functionally active fragment comprises IL-2 region 20-125, wherein the residue positions are in reference to the positions in SEQ ID NO: 84. In some cases, the functionally active fragment comprises IL-2 region 1-130, wherein the residue positions are in reference to the positions in SEQ ID NO: 84. In some cases, the functionally active fragment comprises IL-2 region 1-125, wherein the residue positions are in reference to the positions in SEQ ID NO: 84. In some embodiments, the IL-2 polypeptide is a functionally active fragment. In some cases, the functionally active fragment comprises IL-2 region 10-132, 20-132, 30-132, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, wherein the residue positions are in reference to the positions in SEQ ID NO: 85. In some cases, the functionally active fragment comprises IL-2 region 10-132, wherein the residue positions are in reference to the positions in SEQ ID NO: 85. In some cases, the functionally active fragment comprises IL-2 region 20- 132, wherein the residue positions are in reference to the positions in SEQ ID NO: 85. In some cases, the functionally active fragment comprises IL-2 region 30-132, wherein the residue positions are in reference to the positions in SEQ ID NO: 85. In some cases, the functionally active fragment comprises IL-2 region 10-125, wherein the residue positions are in reference to the positions in SEQ ID NO: 85. In some cases, the functionally active fragment comprises IL-2 region 20-125, wherein the residue positions are in reference to the positions in SEQ ID NO: 85. In some cases, the functionally active fragment comprises IL-2 region 1-130, wherein the residue positions are in reference to the positions in SEQ ID NO: 85. In some cases, the functionally active fragment comprises IL-2 region 1-125, wherein the residue positions are in reference to the positions in SEQ ID NO: 85.

[0431] In some embodiments, described herein is an IL-2 conjugate that comprises an IL-2 polypeptide (e.g., an isolated and purified IL-2 polypeptide, which in some embodiment can include at least one modification) and a conjugating moiety. In some instances, the IL-2 conjugate has a decreased affinity to an IL-2 receptor a (IL-2Ra) subunit relative to a wildtype IL-2 polypeptide. In some cases, the conjugating moiety is bound to an amino acid residue that interacts with IL-2Ra (e.g., at the IL-2 / IL-2Ra interface). In some cases, the conjugating moiety is bound to an amino acid residue that is proximal to the IL-2 / IL-2Ra interface (e.g., about 5 A, about 10 A, about 15 A, or about 20 A away from the IL-2 / IL-2Ra interface). As used herein, the residues involved in the IL-2 / IL-2Ra interface comprise IL-2 residues that form hydrophobic interactions, hydrogen bonds, or ionic interactions with residues from the IL-2Ra subunit.

[0432] In some embodiments, the IL-2 conjugate is a non-alpha IL-2 agonist. As used herein, the term “non-alpha IL-2 agonist” refers to an IL-2 conjugate that reduces the binding of the IL-2 polypeptide to the IL-2 receptor alpha chain (IL2Ra), as compared to a wild-type IL-2 polypeptide, while retaining significant binding to the IL-2 beta-gamma receptors that promote expansion and / or activation of cytotoxic T lymphocytes and / or NK cells. As used herein, the term “significant binding to the IL-2 beta-gamma receptors” refers to a binding affinity that is about 100% (i.e., equivalent), about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, or about 25% of the binding affinity of wild type IL-2 to the IL-2 beta-gamma receptors.

[0433] In some instances, the conjugating moiety is bound to (e.g., in some embodiments covalently attached to) an amino acid residue of an IL-2 polypeptide (e.g., as described herein) selected from an amino acid position Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, F44, Y45, P47, K48, Q57, E60, E61, E62, L63, K64, P65, E68, V69, N71, L72, Q74, S75, K76, N77, M104, C105, E106, Y107, A108, D109, El 10, Ti l l, or Al 12, in which the numbering of the amino acid residues corresponds to SEQ ID NO: 84. In some instances, the amino acid position is selected from Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, F44, Y45, P47, K48, E61, E62, E68, K64, P65, V69, L72, Q74, S75, K76, N77, M104, C105, E106, Y107, A108, D109, El 10, Ti l l, and Al 12. In some instances, the amino acid position is selected from N33, P34, K35, T37, R38, M39, T41, F42, K43, F44, Y45, Q57, E60, E61, E62, L63, K64, P65, E68, V69, N71, L72, M104, C105, E106, Y107, A108, D109, El 10, T111, and Al 12. In some instances, the amino acid position is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some instances, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some instances, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some instances, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some instances, the amino acid position is selected from R38 and K64. In some instances, the amino acid position is selected from E61, E62, and E68. In some cases, the amino acid position is at K35. In some cases, the amino acid position is at T37. In some cases, the amino acid position is at R38. In some cases, the amino acid position is at T41. In some cases, the amino acid position is at F42. In some cases, the amino acid position is at K43. In some cases, the amino acid position is at F44. In some cases, the amino acid position is at Y45. In some cases, the amino acid position is at E61. In some cases, the amino acid position is at E62. In some cases, the amino acid position is at K64. In some cases, the amino acid position is at E68. In some cases, the amino acid position is at P65. In some cases, the amino acid position is at V69. In some cases, the amino acid position is at L72. In some cases, the amino acid position is at Y107. In some cases, the amino acid position is at L72. In some cases, the amino acid position is at DI 09.

[0434] In some instances, the conjugating moiety is bound to (e.g., in some embodiments covalently attached to) an amino acid residue of an IL-2 polypeptide (e.g., as described herein) selected from an amino acid position Y30, K31, N32, P33, K34, T36, R37, T40, F41, K42, F43, Y44, P46, K47, Q56, E59, E60, E61, L62, K63, P64, E67, V68, N70, L71, Q73, S74, K75, N76, M103, C104, E105, Y106, A107, D108, E109, T110, or Al l i, in which the numbering of the amino acid residues corresponds to SEQ ID NO: 85. In some instances, the amino acid position is selected from Y30, K31, N32, P33, K34, T36, R37, T40, F41, K42, F43, Y44, P46, K47, E60, E61, , K63, P64, E67, V68, L71, Q73, S74, K75, N76, M103, C104, E105, Y106, A107, D108, E109, T110, and Al l i. In some instances, the amino acid position is selected from N32, P33, K34, T36, R37, M38, T40, F41, K42, F43, Y44, Q56, E59, E60, E61, L62, K63, P64, E67, V68, N70, L71, M103, C104, E105, Y106, A107, D108, E109, T110, and Al l i. In some instances, the amino acid position is selected from K34, T36, R37, T40, F41, K42, F43, Y44, E60, E61, E67, K63, P64, V68, L71, and Y106. In some instances, the amino acid position is selected from T36, R37, T40, F41, K42, F43, Y44, E60, E61, E67, K63, P64, V68, L71, and Y106. In some instances, the amino acid position is selected from T36, R37, T40, F41, F43, Y44, E60, E61, E67, P64, V68, L71, and Y106. In some instances, the amino acid position is selected from T36, T40, F41, F43, Y44, P64, V68, L71, and Y106. In some instances, the amino acid position is selected from R37 and K63. In some instances, the amino acid position is selected from E60, E61, and E67. In some cases, the amino acid position is at K34. In some cases, the amino acid position is at T36. In some cases, the amino acid position is at R37. In some cases, the amino acid position is at T40. In some cases, the amino acid position is at F41. In some cases, the amino acid position is at K42. In some cases, the amino acid position is at F43. In some cases, the amino acid position is at Y44. In some cases, the amino acid position is at E60. In some cases, the amino acid position is at E61. In some cases, the amino acid position is at K63. In some cases, the amino acid position is at E67. In some cases, the amino acid position is at P64. In some cases, the amino acid position is at V68. In some cases, the amino acid position is at L71. In some cases, the amino acid position is at Y106. In some cases, the amino acid position is at L71. In some cases, the amino acid position is at D108.

[0435] In some instances, the IL-2 conjugate further comprises an additional mutation. In some cases, the additional mutation is at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 in which the numbering of the amino acid residues corresponds to SEQ ID NO: 84. In such cases, the amino acid is conjugated to an additional conjugating moiety for increase in serum half-life, stability, or a combination thereof. Alternatively, the amino acid is first mutated to a natural amino acid such as lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine; or to an unnatural amino acid prior to binding to the additional conjugating moiety.

[0436] In some instances, the IL-2 conjugate further comprises an additional mutation. In some cases, the additional mutation is at an amino acid position selected from K34, T36, R37, T40, F41, K42, F43, Y44, E60, E61, E67, K63, P64, V68, L71, and Y106 in which the numbering of the amino acid residues corresponds to SEQ ID NO: 85. In such cases, the amino acid is conjugated to an additional conjugating moiety for increase in serum half-life, stability, or a combination thereof. Alternatively, the amino acid is first mutated to a natural amino acid such as lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine; or to an unnatural amino acid prior to binding to the additional conjugating moiety.

[0437] In some embodiments, the decreased affinity of the IL-2 polypeptide (e.g., an IL-2 conjugate) to an IL-2 receptor a (IL-2Ra) subunit relative to an equivalent IL-2 polypeptide (e.g., a wild-type IL-2 polypeptide) is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%. In some cases, the decreased affinity is about 10%. In some cases, the decreased affinity is about 20%. In some cases, the decreased affinity is about 40%. In some cases, the decreased affinity is about 50%. In some cases, the decreased affinity is about 60%. In some cases, the decreased affinity is about 80%. In some cases, the decreased affinity is about 90%. In some cases, the decreased affinity is about 99%. In some cases, the decreased affinity is greater than 99%. In some cases, the decreased affinity is about 80%. In some cases, the decreased affinity is 100%.

[0438] In some embodiments, the decreased affinity of the IL-2 polypeptide (e.g., an IL-2 conjugate) to an IL-2 receptor a (IL-2Ra) subunit relative to an equivalent IL-2 polypeptide (e.g., a wild-type IL-2 polypeptide) is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1,000-fold, or more. In some cases, the decreased affinity is about 1-fold. In some cases, the decreased affinity is about 2-fold. In some cases, the decreased affinity is about 4-fold. In some cases, the decreased affinity is about 5-fold. In some cases, the decreased affinity is about 6- fold. In some cases, the decreased affinity is about 8-fold. In some cases, the decreased affinity is about 10-fold. In some cases, the decreased affinity is about 30-fold. In some cases, the decreased affinity is about 50-fold. In some cases, the decreased affinity is about 100-fold. In some cases, the decreased affinity is about 300-fold. In some cases, the decreased affinity is about 500-fold. In some cases, the decreased affinity is about 1000-fold. In some cases, the decreased affinity is more than 1,000-fold.

[0439] In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 85. In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 84.

[0440] In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 85. In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 85.

[0441] In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 86.

[0442] In some embodiments, an IL-2 conjugate described herein is an IL-2 conjugate as described in the International Patent Application No. WO 2019 / 028419, the relevant content of which is incorporated herein for the purposes described herein.

[0443] In some embodiments, an IL-2 conjugate described herein is an IL-2 conjugate as described in the International Patent Application No. WO 2020 / 163532, the relevant content of which is incorporated herein for the purposes described herein.

[0444] Natural and Unnatural Amino Acids

[0445] In some embodiments, an amino acid residue within IL-2 is mutated to lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine prior to binding to (or reacting with) a conjugating moiety. For example, the side chain of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine may bind to a conjugating moiety described herein. In some instances, the amino acid residue is mutated to cysteine, lysine, or histidine. In some cases, the amino acid residue is mutated to cysteine. In some cases, the amino acid residue is mutated to lysine. In some cases, the amino acid residue is mutated to histidine. In some cases, the amino acid residue is mutated to tyrosine. In some cases, the amino acid residue is mutated to tryptophan. In some embodiments, an unnatural amino acid is not conjugated with a conjugating moiety. In some embodiments, a cytokine described herein comprises an unnatural amino acid, wherein the cytokine is conjugated to the protein, wherein the point of attachment is not the unnatural amino acid.

[0446] In some embodiments, an amino acid residue within IL-2 is mutated to an unnatural amino acid prior to binding to a conjugating moiety. In some cases, the mutation to an unnatural amino acid prevents or minimizes a self-antigen response of the immune system. As used herein, the term “unnatural amino acid” or “non-canonical amino acid” refers to an amino acid other than the 20 amino acids that occur naturally in protein. Non-limiting examples of unnatural amino acids include: p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p- methoxyphenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargyl- phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4- propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L- phenylalanine, p-Boronophenylalanine, O-propargyltyrosine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, selenocysteine, p-amino-L- phenylalanine, isopropyl-L-phenylalanine, N6-(2-azidoethoxy)-carbonyl-L-lysine (AzK), an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or a combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and / or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a keto containing amino acid; an amino acid comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an a-hydroxy containing acid; an amino thio acid; an a, a disubstituted amino acid; a P- amino acid; a cyclic amino acid other than proline or histidine, and an aromatic amino acid other than phenylalanine, tyrosine or tryptophan.

[0447] In some embodiments, the unnatural amino acid comprises a selective reactive group, or a reactive group for site-selective labeling of a target polypeptide. In some instances, the chemistry is a biorthogonal reaction (e.g., biocompatible and selective reactions). In some cases, the chemistry is a Cu(I)-catalyzed or “copper-free” alkyne-azide triazole-forming reaction, the Staudinger ligation, inverse-electron-demand Diels-Alder (IEDDA) reaction, “photo-click” chemistry, or a metal-mediated process such as olefin metathesis and Suzuki- Miyaura or Sonogashira cross-coupling.

[0448] In some embodiments, the unnatural amino acid comprises a photoreactive group, which crosslinks, upon irradiation with, e.g., UV.

[0449] In some embodiments, the unnatural amino acid comprises a photo-caged amino acid.

[0450] In some instances, the unnatural amino acid is a para-substituted, meta-substituted, or an ortho-substituted amino acid derivative.

[0451] In some instances, the unnatural amino acid comprises p-acetyl-L-phenylalanine, p- azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, O-methyl-L-tyrosine, p- m ethoxyphenylalanine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2- naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O- acetyl-GlcNAcp-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p- azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, p-amino-L-phenylalanine, or isopropyl-L-phenylalanine.

[0452] In some cases, the unnatural amino acid is 3 -aminotyrosine, 3 -nitrotyrosine, 3,4- dihydroxyphenylalanine, or 3 -iodotyrosine.

[0453] In some cases, the unnatural amino acid is phenyl selenocysteine.

[0454] In some instances, the unnatural amino acid is a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide containing phenylalanine derivative.

[0455] In some instances, the unnatural amino acid is a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide containing lysine derivative.

[0456] In some instances, the unnatural amino acid comprises an aromatic side chain.

[0457] In some instances, the unnatural amino acid does not comprise an aromatic side chain.

[0458] In some instances, the unnatural amino acid comprises an azido group.

[0459] In some instances, the unnatural amino acid comprises a Michael-acceptor group. In some instances, Michael-acceptor groups comprise an unsaturated moiety capable of forming a covalent bond through a 1,2-addition reaction. In some instances, Michael-acceptor groups comprise electron-deficient alkenes or alkynes. In some instances, Michael-acceptor groups include but are not limited to alpha, beta unsaturated: ketones, aldehydes, sulfoxides, sulfones, nitriles, imines, or aromatics.

[0460] In some instances, the unnatural amino acid is dehydroalanine.

[0461] In some instances, the unnatural amino acid comprises an aldehyde or ketone group.

[0462] In some instances, the unnatural amino acid is a lysine derivative comprising an aldehyde or ketone group.

[0463] In some instances, the unnatural amino acid is a lysine derivative comprising one or more O, N, Se, or S atoms at the beta, gamma, or delta position. In some instances, the unnatural amino acid is a lysine derivative comprising O, N, Se, or S atoms at the gamma position.

[0464] In some instances, the unnatural amino acid is a lysine derivative wherein the epsilon N atom is replaced with an oxygen atom.

[0465] In some instances, the unnatural amino acid is a lysine derivative that is not naturally- occurring post-translationally modified lysine.

[0466] In some instances, the unnatural amino acid is an amino acid comprising a side chain, wherein the sixth atom from the alpha position comprises a carbonyl group. In some instances, the unnatural amino acid is an amino acid comprising a side chain, wherein the sixth atom from the alpha position comprises a carbonyl group, and the fifth atom from the alpha position is a nitrogen. In some instances, the unnatural amino acid is an amino acid comprising a side chain, wherein the seventh atom from the alpha position is an oxygen atom.

[0467] In some instances, the unnatural amino acid is a serine derivative comprising selenium. In some instances, the unnatural amino acid is selenoserine (2-amino-3 -hydrosei enopropanoic acid). In some instances, the unnatural amino acid is 2-amino-3 -42-43 -(benzyloxy)-3- oxopropyl)amino)ethyl)selanyl)propanoic acid. In some instances, the unnatural amino acid is 2-amino-3-(phenylselanyl)propanoic acid. In some instances, the unnatural amino acid comprises selenium, wherein oxidation of the selenium results in the formation of an unnatural amino acid comprising an alkene.

[0468] In some instances, the unnatural amino acid comprises a cyclooctynyl group.

[0469] In some instances, the unnatural amino acid comprises a transcycloctenyl group.

[0470] In some instances, the unnatural amino acid comprises a norbornenyl group.

[0471] In some instances, the unnatural amino acid comprises a cyclopropenyl group.

[0472] In some instances, the unnatural amino acid comprises a diazirine group.

[0473] In some instances, the unnatural amino acid comprises a tetrazine group. In some instances, the unnatural amino acid is a lysine derivative, wherein the sidechain nitrogen is carbamylated. In some instances, the unnatural amino acid is a lysine derivative, wherein the side-chain nitrogen is acylated. In some instances, the unnatural amino acid is 2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is 2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is N6-Boc-N6-methyllysine. In some instances, the unnatural amino acid is N6-acetyllysine. In some instances, the unnatural amino acid is pyrrolysine. In some instances, the unnatural amino acid is N6-trifluoroacetyllysine. In some instances, the unnatural amino acid is 2-amino-6-{[(benzyloxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is 2-amino-6-{[(p- iodobenzyloxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is 2-amino-6-{[(p-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is N6-prolyllysine. In some instances, the unnatural amino acid is 2- amino-6-{[(cyclopentyloxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is N6-(cyclopentanecarbonyl)lysine. In some instances, the unnatural amino acid is N6-(tetrahydrofuran-2-carbonyl)lysine. In some instances, the unnatural amino acid is N6-(3- ethynyltetrahydrofuran-2-carbonyl)lysine. In some instances, the unnatural amino acid is N6- ((prop-2-yn-l-yloxy)carbonyl)lysine. In some instances, the unnatural amino acid is 2-amino- 6-{[(2-azidocyclopentyloxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is N6-(2-azidoethoxy)-carbonyl-lysine. In some instances, the unnatural amino acid is 2-amino-6-{[(2-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is 2-amino-6-{[(2-cyclooctynyloxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is N6-(2-aminobut-3-ynoyl)lysine. In some instances, the unnatural amino acid is 2-amino-6-((2-aminobut-3-ynoyl)oxy)hexanoic acid. In some instances, the unnatural amino acid is N6-(allyloxycarbonyl)lysine. In some instances, the unnatural amino acid is N6-(butenyl-4-oxycarbonyl)lysine. In some instances, the unnatural amino acid is N6-(pentenyl-5-oxycarbonyl)lysine. In some instances, the unnatural amino acid is N6-((but-3-yn-l-yloxy)carbonyl)-lysine. In some instances, the unnatural amino acid is N6- ((pent-4-yn-l-yloxy)carbonyl)-lysine. In some instances, the unnatural amino acid is N6- (thiazolidine-4-carbonyl)lysine. In some instances, the unnatural amino acid is 2-amino-8- oxononanoic acid. In some instances, the unnatural amino acid is 2-amino-8-oxooctanoic acid. In some instances, the unnatural amino acid is N6-(2-oxoacetyl)lysine. In some instances, the unnatural amino acid is N6-propionyllysine. In some instances, the unnatural amino acid is N6-butyryllysine, In some instances, the unnatural amino acid is N6-(but-2-enoyl)lysine, In some instances, the unnatural amino acid is N6- ((bicyclo[2.2.1]hept-5-en-2-yloxy)carbonyl)lysine. In some instances, the unnatural amino acid is N6-((spiro[2.3]hex-l-en-5-ylmethoxy)carbonyl)lysine. In some instances, the unnatural amino acid is N6-(((4-(l-(trifluoromethyl)cycloprop-2-en-l-yl)benzyl)oxy)carbonyl)lysine. In some instances, the unnatural amino acid is N6-((bicyclo[2.2.1]hept-5-en-2- ylmethoxy)carbonyl)lysine. In some instances, the unnatural amino acid is cysteinyllysine. In some instances, the unnatural amino acid is N6-((l-(6-nitrobenzo[d][l,3]dioxol-5- yl)ethoxy)carbonyl)lysine. In some instances, the unnatural amino acid is N6-((2-(3-methyl- 3H-diazirin-3-yl)ethoxy)carbonyl)lysine. In some instances, the unnatural amino acid is N6- ((3-(3-methyl-3H-diazirin-3-yl)propoxy)carbonyl)lysine. In some instances, the unnatural amino acid is N6-((meta nitrobenzyloxy)N6-methylcarbonyl)lysine. In some instances, the unnatural amino acid is N6-((bicyclo[6.1.0]non-4-yn-9-ylmethoxy)carbonyl)-lysine. In some instances, the unnatural amino acid is N6-((cyclohept-3-en-l-yloxy)carbonyl)-L-lysine.

[0474] In some instances, the unnatural amino acid is 2-amino-3- (((((benzyloxy)carbonyl)amino)methyl)selanyl)propanoic acid.

[0475] Conjugating Moi eties - Water-Soluble Polymers

[0476] In some embodiments, a conjugating moiety described herein is a water-soluble polymer. In some instances, the water-soluble polymer is a nonpeptidic, nontoxic, and biocompatible. As used herein, a substance is considered biocompatible if the beneficial effects associated with use of the substance alone or with another substance (e.g., an active agent such as a cytokine moiety) in connection with living tissues (e.g., administration to a patient) outweighs any deleterious effects as evaluated by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist. In some instances, a water-soluble polymer is further non- immunogenic. In some instances, a substance is considered non-immunogenic if the intended use of the substance in vivo does not produce an undesired immune response (e.g., the formation of antibodies) or, if an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist.

[0477] In some instances, the water-soluble polymer is characterized as having from about 2 to about 300 termini. Exemplary water soluble polymers include, but are not limited to, poly(alkylene glycols) such as polyethylene glycol (“PEG”), polypropylene glycol) (“PPG”), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefinic alcohol), polyvinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polydimethylacrylamide (PDAAm), polyphosphazene, polyoxazolines (“POZ”) (which are described in WO 2008 / 106186), poly(N-acryloylmorpholine), and combinations of any of the foregoing.

[0478] In some cases, the water-soluble polymer is not limited to a particular structure. In some cases, the water-soluble polymer is linear (e.g., an end capped, e.g., alkoxy PEG or a bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), a dendritic (or star) architecture, each with or without one or more degradable linkages. Moreover, the internal structure of the water-soluble polymer can be organized in any number of different repeat patterns and can be selected from the group consisting of homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer.

[0479] In some instances, the water-soluble polymer is represented by a length of repeating polymeric units, for example, a number n of polyethylene glyocol units. In some instances, the water-soluble polymer has the structure: wherein the wavy line indicates attachment to a linker, reactive group, or unnatural amino acid, and n is 1-5000. In some instances, the water-soluble polymer has the structure: wherein the wavy line indicates attachment to a linker, reactive group, or unnatural amino acid, “Cap” indicates a capping group (for example, such as — OCH3, — O(Ci-Ce alkyl), — SMe, — S(Ci-C6alkyl), — CO2H, — CO2(Ci-C6alkyl), — CONH2, — CONH(CI-C6alkyl), — CON(Ci- Ce alkyl )2, — NH2, — SH, or OH) and n is 1-5000. In some embodiments, n is 100-2000, 200- 1000, 300-750, 400-600, 450-550, 400-2000, 750-3000, or 100-750. In some embodiments, n is about 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000. In some embodiments, n is at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or at least 1000. In some embodiments, n is no more than 100, 200, 300, 400, 500, 600, 700, 800, 900, or no more than 1000. In some embodiments, the n is represented as an average length of the water-soluble polymer.

[0480] In some embodiments, the weight-average molecular weight of the water-soluble polymer in the IL-2 conjugate is from about 100 Daltons to about 150,000 Daltons. Exemplary ranges include, for example, weight-average molecular weights in the range of greater than 5,000 Daltons to about 100,000 Daltons, in the range of from about 6,000 Daltons to about 90,000 Daltons, in the range of from about 10,000 Daltons to about 85,000 Daltons, in the range of greater than 10,000 Daltons to about 85,000 Daltons, in the range of from about 20,000 Daltons to about 85,000 Daltons, in the range of from about 53,000 Daltons to about 85,000 Daltons, in the range of from about 25,000 Daltons to about 120,000 Daltons, in the range of from about 29,000 Daltons to about 120,000 Daltons, in the range of from about 35,000 Daltons to about 120,000 Daltons, and in the range of from about 40,000 Daltons to about 120,000 Daltons.

[0481] Exemplary weight-average molecular weights for the water-soluble polymer include about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1,000 Daltons, about 1,500 Daltons, about 2,000 Daltons, about 2,200 Daltons, about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about 4,400 Daltons, about 4,500 Daltons, about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000

[0482] Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000

[0483] Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000

[0484] Daltons, about 50,000 Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000

[0485] Daltons, about 70,000 Daltons, and about 75,000 Daltons. Branched versions of the water- soluble polymer (e.g., a branched 40,000 Dalton water-soluble polymer comprised of two 20,000 Dalton polymers) having a total molecular weight of any of the foregoing can also be used. In one or more embodiments, the conjugate will not have any PEG moieties attached, either directly or indirectly, with a PEG having a weight average molecular weight of less than about 6,000 Daltons.

[0486] PEGs will typically comprise a number of (OCH2CH2) monomers [or (CH2CH2O) monomers, depending on how the PEG is defined]. As used herein, the number of repeating units is identified by the subscript “n” in “(OCEECEEjn.” Thus, the value of (n) typically falls within one or more of the following ranges: from 2 to about 3400, from about 100 to about 2300, from about 100 to about 2270, from about 136 to about 2050, from about 225 to about 1930, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1,200 to about 1,900. For any given polymer in which the molecular weight is known, it is possible to determine the number of repeating units (i.e., “n”) by dividing the total weight-average molecular weight of the polymer by the molecular weight of the repeating monomer.

[0487] In some instances, the water-soluble polymer is an end-capped polymer, that is, a polymer having at least one terminus capped with a relatively inert group, such as a lower Ci- 6 alkoxy group, or a hydroxyl group. When the polymer is PEG, for example, a methoxy-PEG (commonly referred to as mPEG) may be used, which is a linear form of PEG wherein one terminus of the polymer is a methoxy ( — OCH3) group, while the other terminus is a hydroxyl or other functional group that can be optionally chemically modified.

[0488] In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a linear or branched PEG group. In some embodiments, the PEG group is a linear PEG group. In some embodiments, the PEG group is a branched PEG group. In some embodiments, the PEG group is a methoxy PEG group. In some embodiments, the PEG group is a linear or branched methoxy PEG group. In some embodiments, the PEG group is a linear methoxy PEG group. In some embodiments, the PEG group is a branched methoxy PEG group. In some embodiments, the PEG group is a linear or branched PEG group having an average molecular weight of from about 100 Daltons to about 150,000 Daltons. Exemplary ranges include, for example, weight-average molecular weights in the range of greater than 5,000 Daltons to about 100,000 Daltons, in the range of from about 6,000 Daltons to about 90,000 Daltons, in the range of from about 10,000 Daltons to about 85,000 Daltons, in the range of greater than 10,000 Daltons to about 85,000 Daltons, in the range of from about 20,000 Daltons to about 85,000 Daltons, in the range of from about 53,000 Daltons to about 85,000 Daltons, in the range of from about 25,000 Daltons to about 120,000 Daltons, in the range of from about 29,000 Daltons to about 120,000 Daltons, in the range of from about 35,000 Daltons to about 120,000 Daltons, and in the range of from about 40,000 Daltons to about 120,000 Daltons. Exemplary weightaverage molecular weights for the PEG group include about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1,000 Daltons, about 1,500 Daltons, about 2,000 Daltons, about 2,200 Daltons, about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about 4,400 Daltons, about 4,500 Daltons, about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000

[0489] Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000

[0490] Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000

[0491] Daltons, about 60,000 Daltons, about 65,000 Daltons, about 70,000 Daltons, about 75,000

[0492] Daltons, about 80,000 Daltons, about 90,000 Daltons, about 95,000 Daltons, and about 100,000 Daltons. In some embodiments, the PEG group is a linear PEG group having an average molecular weight as disclosed above. In some embodiments, the PEG group is a branched PEG group having an average molecular weight as disclosed above. In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a linear or branched PEG group having a defined molecular weight ±10%, or 15% or 20% or 25%. For example, included within the scope of the present disclosure are IL-2 conjugates comprising a PEG group having a molecular weight of 30,000 Da±3000 Da, or 30,000 Da±4,500 Da, or 30,000 Da±6,000 Da.

[0493] In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a linear or branched PEG group having an average molecular weight of from about 5,000 Daltons to about 60,000 Daltons. In some embodiments, the PEG group is a linear or branched PEG group having an average molecular weight of about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about

[0494] 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about

[0495] 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000 Daltons, about

[0496] 60,000 Daltons, about 65,000 Daltons, about 70,000 Daltons, about 75,000 Daltons, about

[0497] 80,000 Daltons, about 90,000 Daltons, about 95,000 Daltons, and about 100,000 Daltons. In some embodiments, the PEG group is a linear or branched PEG group having an average molecular weight of about 5,000 Daltons, about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a linear or branched PEG group having an average molecular weight of about 5,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a linear PEG group having an average molecular of about 5,000 Daltons, about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a branched PEG group having an average molecular weight of about 5,000 Daltons, about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons.

[0498] In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a linear methoxy PEG group having an average molecular weight of from about 5,000 Daltons to about 60,000 Daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about

[0499] 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about

[0500] 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000 Daltons, about

[0501] 60,000 Daltons, about 65,000 Daltons, about 70,000 Daltons, about 75,000 Daltons, about

[0502] 80,000 Daltons, about 90,000 Daltons, about 95,000 Daltons, and about 100,000 Daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 5,000 Daltons, about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 5,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular of about 5,000 Daltons, about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular of about 5,000 Daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular of about 10,000 Daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular of about 20,000 Daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular of about 30,000 Daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular of about 50,000 Daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular of about 60,000 Daltons. In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a linear methoxy PEG group having a defined molecular weight ±10%, or 15% or 20% or 25%. For example, included within the scope of the present disclosure are IL-2 conjugates comprising a linear methoxy PEG group having a molecular weight of 30,000 Da±3000 Da, or 30,000 Da±4,500 Da, or 30,000 Da±6,000 Da.

[0503] In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a branched methoxy PEG group having an average molecular weight of from about 5,000 Daltons to about 60,000 Daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about

[0504] 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about

[0505] 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000 Daltons, about

[0506] 60,000 Daltons, about 65,000 Daltons, about 70,000 Daltons, about 75,000 Daltons, about

[0507] 80,000 Daltons, about 90,000 Daltons, about 95,000 Daltons, and about 100,000 Daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5,000 Daltons, about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular of about 5,000 Daltons, about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular of about 5,000 Daltons, about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular of about 5,000 Daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular of about 10,000 Daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular of about 20,000 Daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular of about 30,000 Daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular of about 50,000 Daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular of about 60,000 Daltons. In some embodiments, the PEG group comprising the IL- 2 conjugates disclosed herein is a branched methoxy PEG group having a defined molecular weight ±10%, or 15% or 20% or 25%. For example, included within the scope of the present disclosure are IL-2 conjugates comprising a branched methoxy PEG group having a molecular weight of 30,000 Da±3000 Da, or 30,000 Da±4,500 Da, or 30,000 Da±6,000 Da.

[0508] In some embodiments, exemplary water-soluble polymers include, but are not limited to, linear or branched discrete PEG (dPEG) from Quanta Biodesign, Ltd; linear, branched, or forked PEGs from Nektar Therapeutics; and Y-shaped PEG derivatives from JenKem Technology.

[0509] In some embodiments, an IL-2 polypeptide described herein is conjugated to a water- soluble polymer selected from poly(alkylene glycols) such as polyethylene glycol (“PEG”), polypropylene glycol) (“PPG”), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), polydimethylacrylamide (PDAAm), poly(N-(2 -hydroxypropyl) methacrylamide) (PHPMA), polyphosphazene, polyoxazolines (“POZ”), poly(N-acryloylmorpholine), and a combination thereof. In some instances, the cytokine polypeptide is conjugated to PEG (e.g., PEGylated). In some instances, the cytokine polypeptide is conjugated to PPG. In some instances, the cytokine polypeptide is conjugated to POZ. In some instances, the cytokine polypeptide is conjugated to PVP. In some instances, a water-soluble polymer comprises a polyglycerol (PG). In some cases, the polyglycerol is a hyperbranched PG (HPG) (e.g., as described by Imran, et al. “Influence of architecture of high molecular weight linear and branched polyglycerols on their biocompatibility and biodistribution,” Biomaterials 33:9135-9147 (2012)). In other cases, the polyglycerol is a linear PG (LPG). In additional cases, the polyglycerol is a midfunctional PG, a linear-block-hyperbranched PG (e.g., as described by Wurm et. Al., “Squaric acid mediated synthesis and biological activity of a library of linear and hyperbranched poly(glycerol)-protein conjugates,” Biomacromolecules 13: 1161-1171 (2012)), or a side-chain functional PG (e.g., as described by Li, et. al., “Synthesis of linear polyether polyol derivatives as new materials for bioconjugation,” Bioconjugate Chem. 20:780-789 (2009).

[0510] In some embodiments, a water-soluble polymer is a degradable synthetic PEG alternative. Exemplary degradable synthetic PEG alternatives include, but are not limited to, poly[oligo(ethylene glycol)methyl methacrylate] (POEGMA); backbone modified PEG derivatives generated by polymerization of telechelic, or di-end-functionalized PEG-based macromonomers; PEG derivatives comprising comonomers comprising degradable linkage such as poly[(ethylene oxie)-co-(methylene ethylene oxide)][P(EO-co-MEO)], cyclic ketene acetals such as 5,6-benzo-2-methylene-l,3-dioxepane (BMDO), 2-methylene-l,3-dioxepane (MDO), and 2-methylene-4-phenyl-l,3-dioxolane (MPDL) copolymerized with OEGMA; or poly-(s-caprolactone)-graft-poly(ethylene oxide) (PCL-g-PEO).

[0511] In some cases, the IL-2 polypeptide is conjugated to a degradable synthetic PEG alternative, such as for example, POEGM; backbone modified PEG derivatives generated by polymerization of telechelic, or di-end-functionalized PEG-based macromonomers; P(EO-co- MEO); cyclic ketene acetals such as BMDO, MDO, and MPDL copolymerized with OEGMA; or PCL-g-PEO.

[0512] In some embodiments, a water-soluble polymer comprises a poly(zwitterions). Exemplary poly(zwitterions) include, but are not limited to, poly(sulfobetaine methacrylate) (PSBMA), poly(carboxybetaine methacrylate) (PCBMA), and poly(2-methyacryloyloxyethyl phosphorylcholine) (PMPC).

[0513] In some embodiments, a water-soluble polymer comprises a polycarbonate. Exemplary polycarbones include, but are not limited to, pentafluorophenyl 5-methyl-2-oxo-l,3-dioxane- 5-carboxylate (MTC-OC6F5).

[0514] In some embodiments, a water-soluble polymer comprises a polymer hybrid, such as for example, a polycarbonate / PEG polymer hybrid, a peptide / protein-polymer conjugate, or a hydroxylcontaining and / or zwitterionic derivatized polymer (e.g., a hydroxylcontaining and / or zwitterionic derivatized PEG polymer).

[0515] In some instances, a water-soluble polymer comprises a polysaccharide. Exemplary polysaccharides include, but are not limited to, dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES).

[0516] In some cases, a water-soluble polymer comprises a glycan. Exemplary classes of glycans include N-linked glycans, O-linked glycans, glycolipids, O-GlcNAc, and glycosaminoglycans.

[0517] In some embodiments, a water-soluble polymer comprises a polyoxazoline polymer. A polyoxazoline polymer is a linear synthetic polymer, and similar to PEG, comprises a low polydispersity. In some instances, a polyoxazoline polymer is a polydispersed polyoxazoline polymer, characterized with an average molecule weight. In some cases, the average molecule weight of a polyoxazoline polymer includes, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, 100,000, 200,000, 300,000, 400,000, or 500,000 Da. In some instances, a polyoxazoline polymer comprises poly(2-methyl 2-oxazoline) (PMOZ), poly(2- ethyl 2-oxazoline) (PEOZ), or poly(2-propyl 2-oxazoline) (PPOZ).

[0518] In some instances, a water-soluble polymer comprises a polyacrylic acid polymer.

[0519] In some instances, a water-soluble polymer comprises polyamine. Polyamine is an organic polymer comprising two or more primary amino groups. In some embodiments, a polyamine includes a branched polyamine, a linear polyamine, or cyclic polyamine. In some cases, a polyamine is a low-molecular-weight linear polyamine. Exemplary polyamines include putrescine, cadaverine, spermidine, spermine, ethylene diamine, 1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, and piperazine.

[0520] In some instances, a water-soluble polymer is described in U.S. Pat. Nos. 7,744,861, 8,273,833, and 7,803,777.

[0521] Conjugating Moi eties - Lipids

[0522] In some embodiments, a conjugating moiety described herein is a lipid. In some instances, the lipid is a fatty acid. In some cases, the fatty acid is a saturated fatty acid. In other cases, the fatty acid is an unsaturated fatty acid. Exemplary fatty acids include, but are not limited to, fatty acids comprising from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms. In some cases, the lipid binds to one or more serum proteins, thereby increasing serum stability and / or serum half-life.

[0523] In some cases, the fatty acid comprises about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbon atoms in length. In some cases, the fatty acid comprises caproic acid (hexanoic acid), enanthic acid (heptanoic acid), caprylic acid (octanoic acid), pelargonic acid (nonanoic acid), capric acid (decanoic acid), undecylic acid (undecanoic acid), lauric acid (dodecanoic acid), tridecylic acid (tridecanoic acid), myristic acid (tetradecanoic acid), pentadecylic acid (pentadecanoic acid), palmitic acid (hexadecanoic acid), margaric acid (heptadecanoic acid), stearic acid (octadecanoic acid), nonadecylic acid (nonadecanoic acid), arachidic acid (eicosanoic acid), heneicosylic acid (heneicosanoic acid), behenic acid (docosanoic acid), tricosylic acid (tricosanoic acid), lignoceric acid (tetracosanoic acid), pentacosylic acid (pentacosanoic acid), or cerotic acid (hexacosanoic acid).

[0524] Conjugating Moi eties - Proteins

[0525] In some embodiments, a conjugating moiety described herein is a protein or a binding fragment thereof. Exemplary proteins include albumin, transferrin, or transthyretin. In some instances, the protein or a binding fragment thereof comprises an antibody, or its binding fragments thereof. In some cases, a cytokine conjugate comprises a protein or a binding fragment thereof. In some cases, an IL-2 conjugate comprising a protein or a binding fragment thereof has a reduced IL-2 interaction with one or more IL-2R subunits. In additional cases, the protein or a binding fragment thereof blocks IL-2 interaction with one or more IL-2R subunits.

[0526] In some embodiments, the conjugating moiety is albumin. Albumin is a family of water- soluble globular proteins. It is commonly found in blood plasma, comprising about 55-60% of all plasma proteins. Human serum albumin (HSA) is a 585 amino acid polypeptide in which the tertiary structure is divided into three domains, domain I (amino acid residues 1-195), domain II (amino acid residues 196-383), and domain III (amino acid residues 384-585). Each domain further comprises a binding site, which can interact either reversibly or irreversibly with endogenous ligands such as long- and medium-chain fatty acids, bilirubin, or hemin, or exogenous compounds such as heterocyclic or aromatic compounds. In some embodiments, the conjugating moiety is transferrin. Transferrin is a 679 amino acid polypeptide that is about 80 kDa in size and comprises two Fe<3+> binding sites with one at the N-terminal domain and the other at the C-terminal domain. In some instances, human transferrin has a half-life of about 7-12 days.

[0527] Conjugation

[0528] Linkers

[0529] In some embodiments, useful functional reactive groups for conjugating or binding a conjugating moiety to an IL-2 polypeptide described herein include, for example, zero or higher-order linkers. In some instances, an unnatural amino acid incorporated into an interleukin described herein comprises a functional reactive group. In some instances, a linker comprises a functional reactive group that reacts with an unnatural amino acid incorporated into an interleukin described herein. In some instances, a conjugating moiety comprises a functional reactive group that reacts with an unnatural amino acid incorporated into an interleukin described herein. In some instances, a conjugating moiety comprises a functional reactive group that reacts with a linker (optionally pre-attached to a cytokine peptide) described herein. In some embodiments, a linker comprises a reactive group that reacts with a natural amino acid in a cytokine peptide described herein. In some cases, higher-order linkers comprise bifunctional linkers, such as homobifunctional linkers or heterobifunctional linkers. Exemplary homobifuctional linkers include, but are not limited to, Lomant's reagent dithiobis (succinimidylpropionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propri onate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl- 3,3'-dithiobispropionimidate (DTBP), l,4-di-3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), such as e.g. l,5-difluoro-2,4-dinitrobenzene or l,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'- dinitrophenylsulfone (DFDNPS), bis-[P-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3 '-dimethylbenzidine, benzidine, a,a'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene- bi s(i odoacetami de) . In some embodiments, the bifunctional linker comprises a heterobifunctional linker. Exemplary heterobifunctional linker include, but are not limited to, amine-reactive and sulfhydryl cross-linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long- chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chain N- succinimidyl 3 -(2 -pyridyldithio) propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-a- methyl-a-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[a-methyl-a-(2- pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate (sMCC), sulfosuccinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N- hydroxy succinimide ester (MBs), m-maleimidobenzoyl-N-hydroxy sulfosuccinimide ester (sulfo-MBs), N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB), sulfosuccinimidyl(4- iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate

[0530] (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(y- maleimidobutyryloxy)succinimide ester (GMBs), N-(y- maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs), succinimidyl 6- ((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-

[0531] (((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4- (((iodoacetyl)amino)methyl)cyclohexane-l -carboxylate (sIAC), succinimidyl 6-((((4- iodoacetyl)amino)methyl)cyclohexane-l-carbonyl)amino) hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl -reactive and sulfhydryl-reactive cross-linkers such as 4-(4-N- maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-l- carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), aminereactive and photoreactive cross-linkers such as N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl- 2-(p-azidosalicylamido)ethyl-l, 3 '-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4- azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N- succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'- azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2- nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)- ethyl- 1 ,3 '-di thiopropionate (sAND), N-succinimidyl-4(4-azidophenyl) 1 ,3 '-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)- 1,3 '-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4- methylcoumarin-3-acetamide)ethyl- 1,3 '-dithiopropionate (sAED), sulfosuccinimidyl 7-azido- 4-methylcoumain-3 -acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p- nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive cross-linkers such as l-(p-Azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3 '-(2'-pyridyldithio)propionamide (APDP), benzophenone- 4-iodoacetamide, benzophenone-4-maleimide carbonyl-reactive and photoreactive crosslinkers such as p-azidobenzoyl hydrazide (ABH), carboxylate-reactive and photoreactive cross-linkers such as 4-(p-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive cross-linkers such as p-azidophenyl glyoxal (APG).

[0532] In some instances, the reactive functional group comprises a nucleophilic group that is reactive to an electrophilic group present on a conjugating moiety or on IL-2. Exemplary electrophilic groups include carbonyl groups-such as aldehyde, ketone, carboxylic acid, ester, amide, enone, acyl halide or acid anhydride. In some embodiments, the reactive functional group is aldehyde. Exemplary nucleophilic groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In some embodiments, an unnatural amino acid incorporated into an interleukin described herein comprises an electrophilic group.

[0533] In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is a dipeptide linker. In some embodiments, the dipeptide linker is valinecitrulline (Val-Cit), phenylalanine-lysine (Phe-Lys), valine-alanine (Vai-Ala) and valine-lysine (Val-Lys). In some embodiments, the dipeptide linker is valine-citrulline.

[0534] In some embodiments, the linker is a peptide linker comprising, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids. In some instances, the peptide linker comprises at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or less amino acids. In additional cases, the peptide linker comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.

[0535] In some embodiments, the linker comprises a self-immolative linker moiety. In some embodiments, the self-immolative linker moiety comprises p-aminobenzyl alcohol (PAB), p- aminobenzyoxy carbonyl (PABC), or derivatives or analogs thereof. In some embodiments, the linker comprises a dipeptide linker moiety and a self-immolative linker moiety. In some embodiments, the self-immolative linker moiety is such as described in U.S. Pat. No. 9,089,614 and WIPO Application No. WO2015038426. In some embodiments, the cleavable linker is glucuronide. In some embodiments, the cleavable linker is an acid-cleavable linker. In some embodiments, the acid-cleavable linker is hydrazine. In some embodiments, the cleavable linker is a reducible linker.

[0536] In some embodiments, the linker comprises a maleimide group. In some instances, the maleimide group is also referred to as a maleimide spacer. In some instances, the maleimide group further comprises a caproic acid, forming maleimidocaproyl (me). In some cases, the linker comprises maleimidocaproyl (me). In some cases, linker is maleimidocaproyl (me). In other instances, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate (sulfo-sMCC) described above.

[0537] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some instances, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of tiosuccinimide ring hydrolysis, thereby eliminating maleimide from undergoing an elimination reaction through a retro-Michael reaction. In some instances, the self-stabilizing maleimide is a maleimide group described in Lyon, et al., “Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat. Biotechnol. 32(10): 1059-1062 (2014). In some instances, the linker comprises a self-stabilizing maleimide. In some instances, the linker is a self-stabilizing maleimide.

[0538] Conjugation Chemistry

[0539] Various conjugation reactions are used to conjugate linkers, conjugation moieties, and unnatural amino acids incorporated into the IL-2 polypeptides described herein. Such conjugation reactions are often compatible with aqueous conditions, such as “bioorthogonal” reactions. In some embodiments, conjugation reactions are mediated by chemical reagents such as catalysts, light, or reactive chemical groups found on linkers, conjugation moieties, or unnatural amino acids. In some embodiments, conjugation reactions are mediated by enzymes. In some embodiments, a conjugation reaction used herein is described in Gong, Y., Pan, L. Tett. Lett. 2015, 56, 2123. In some embodiments, a conjugation reaction used herein is described in Chen, X.; Wu. Y-W. Org. Biomol. Chem. 2016, 14, 5417.

[0540] In some embodiments described herein, a conjugation reaction comprises reaction of a ketone or aldehyde with a nucleophile. In some embodiments, a conjugation reaction comprises reaction of a ketone with an aminoxy group to form an oxime. In some embodiments, a conjugation reaction comprises reaction of a ketone with an aryl or heteroaryl amine group to form an imine. In some embodiments, a conjugation reaction comprises reaction of an aldehyde with an aryl or heteroaryl amine group to form an imine.

[0541] In some embodiments, a conjugation reaction described herein results in cytokine peptide comprising a linker or conjugation moiety attached via an oxime. In some embodiments, a conjugation reaction comprises a Pictet-Spengler reaction of an aldehyde or ketone with a tryptamine nucleophile. In some embodiments, a conjugation reaction comprises a hydrazino-Pictet-Spengler reaction. In some embodiments, a conjugation reaction comprises a Pictet-Spengler ligation.

[0542] In some embodiments described herein, a conjugation reaction described herein comprises reaction of an azide and a phosphine (Staudinger ligation). In some embodiments, the phosphine is an aryl phosphine. In some embodiments, the aryl phosphine comprises an ortho ester group. In some embodiments, the phosphine comprises the structure methyl 2- (diphenylphosphaneyl)benzoate. In some embodiments, a conjugation reaction described herein results in cytokine peptide comprising a linker or conjugation moiety attached via an arylamide. In some embodiments, a conjugation reaction described herein results in cytokine peptide comprising a linker or conjugation moiety attached via an amide.

[0543] In some embodiments described herein, a conjugation reaction described herein comprises a 1,3-dipolar cycloaddition reaction. In some embodiments, the 1,3-dipolar cycloaddition reaction comprises reaction of an azide and a phosphine (“Click” reaction). In some embodiments, the conjugation reaction is catalyzed by copper. In some embodiments, a conjugation reaction described herein results in cytokine peptide comprising a linker or conjugation moiety attached via a triazole. In some embodiments, a conjugation reaction described herein comprises reaction of an azide with a strained olefin. In some embodiments, a conjugation reaction described herein comprises reaction of an azide with a strained alkyne. In some embodiments, a conjugation reaction described herein comprises reaction of an azide with a cycloalkyne, for example, OCT, DIFO, DIFBO, DIBO, BARAC, TMTH, or other strained cycloalkyne, the structures of which are shown in Gong, Y., Pan, L. Tett. Lett. 2015, 56, 2123. In some embodiments, a 1,3-dipolar cycloaddition reaction is catalyzed by light (“photoclick”). In some embodiments, a conjugation reaction described herein comprises reaction of a terminal allyl group with a tetrazole and light. In some embodiments, a conjugation reaction described herein comprises reaction of a terminal alkynyl group with a tetrazole and light. In some embodiments, a conjugation reaction described herein comprises reaction of an O-allyl amino acid with a tetrazine and light. In some embodiments, a conjugation reaction described herein comprises reaction of O-allyl tyrosine with a tetrazine and light. In some embodiments described herein, a conjugation reaction described herein comprises are cycloaddition reaction between an azide moiety, such as that contained in a protein containing an amino acid residue derived from N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and a strained cycloalkyne, such as that derived from DBCO, which is a chemical moiety comprising a dibenzocyclooctyne group. PEG groups comprising a DBCO moiety are commercially available or may be prepared by methods known to those of ordinary skill in the art.

[0544] Conjugation reactions such as a click reaction described herein may generate a single regioisomer, or a mixture of regioisomers. In some instances the ratio of regioisomers is about 1 : 1. In some instances the ratio of regioisomers is about 2:1. In some instances the ratio of regioisomers is about 1.5 : 1. In some instances the ratio of regioisomers is about 1.2: 1. In some instances the ratio of regioisomers is about 1.1 : 1. In some instances the ratio of regioisomers is greater than 1 : 1.

[0545] In some embodiments, an IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 84 or an amino acid sequence with about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 84, wherein the amino acid at position P65 is replaced by the structure of Formula (I):

[0546] W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; q is 1, 2, or 3; X has the structure: wherein X-l indicates the point of attachment to the preceding amino acid residue; and wherein X+l indicates the point of attachment to the following amino acid residue.

[0547] In some embodiments, the PEG group has an average molecular weight of about 30 kDa.

[0548] In some embodiments, the

[0549] In some embodiments, the

[0550] In some embodiments, the

[0551] In some embodiments, an IL-2 conjugate is provided as a structure of Formula (IV): wherein: q is 1, 2, or 3;

[0552] X has the structure: wherein X-l indicates the point of attachment to the preceding amino acid residue; and wherein X+l indicates the point of attachment to the following amino acid residue.

[0553] In some embodiments, an IL-2 conjugate is provided as a structure of Formula (V):

[0554] Formula (V); wherein: q is 1, 2, or 3;

[0555] X has the structure: wherein X-l indicates the point of attachment to the preceding amino acid residue; and wherein X+l indicates the point of attachment to the following amino acid residue. In some embodiments, an IL-2 conjugate is provided as a mixture comprising (i) IL-2 conjugates in which the structure of Formula (I) has the structure of Formula (IV), and (ii) IL- 2 conjugates in which the structure of Formula (I) has the structure of Formula (V):

[0556] Formula (V); wherein: q is 1, 2, or 3; X has the structure: wherein X-l indicates the point of attachment to the preceding amino acid residue; and wherein X+l indicates the point of attachment to the following amino acid residue.

[0557] In some embodiments, an IL-2 conjugate is provided as a structure of Formula (XII):

[0558] Formula (XII); wherein: n is an integer such that a PEG group having the structure of -(OCFECtEJn-OCHs has a molecular weight of about 30 kDa; q is 1, 2, or 3; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 84 that are not replaced. In some embodiments, the q is 1. In some embodiments, the q is 2. In some embodiments, the q is 3.

[0559] In some embodiments, an IL-2 conjugate is provided as a structure of Formula (XIII):

[0560] Formula (XIII); wherein: n is an integer such that a PEG group having the structure of -(OCEECEEjn-OCIL has a molecular weight of about 30 kDa; q is 1, 2, or 3; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 84 that are not replaced. In some embodiments, the q is 1. In some embodiments, the q is 2. In some embodiments, the q is 3.

[0561] In some embodiments, an IL-2 conjugate is provided as a mixture comprising (i) IL-2 conjugates in which the structure of Formula (I) has the structure of Formula (XII), and (ii) IL- 2 conjugates in which the structure of Formula (I) has the structure of Formula (XIII):

[0562] Formula (XII);

[0563] Formula (XIII); wherein: n is an integer such that a PEG group having the structure of -(OCEECEEjn-OCIL has a molecular weight of about 30 kDa; q is 1, 2, or 3; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 84 that are not replaced. In some embodiments, the q is 1. In some embodiments, the q is 2. In some embodiments, the q is 3.

[0564] Exemplary IL-2 Conjugate Embodiments

[0565] 1. An IL-2 conjugate comprising: an IL-2 polypeptide comprising a substituted lysine comprising an axidoethoxy moiety covalently attached to a conjugating moiety comprising a polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 84; and the substituted lysine substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO: 84.

[0566] 2. The IL-2 conjugate of embodiment 1, which is a pharmaceutically acceptable salt, solvate, or hydrate.

[0567] 3. The IL-2 conjugate of embodiment 1 or 2, wherein the PEG has a molecular weight of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, or 50 kDa. 4. The IL-2 conjugate of any one of embodiments 1-3, wherein the substituted lysine substitutes for the amino acid at position P65 in reference to the amino acid positions within SEQ ID NO: 84.

[0568] 5. The IL-2 conjugate of any one of embodiments 1-4, wherein the IL-2 polypeptide comprises an N-terminal deletion of one residue relative to SEQ ID NO: 84.

[0569] 6. The IL-2 conjugate of embodiment 4, wherein the PEG has a molecular weight of about 30 kDa.

[0570] 7. The IL-2 conjugate of any one of embodiments 1-6, wherein the substituted lysine is N6- ((2-azidoethoxy)-carbonyl)-L-lysine.

[0571] 8. An IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 124, wherein [AzK_Ll_PEG30 kD] is an L-amino acid having the structure of Formula (XVI) or Formula (XVII):

[0572] (Formula XVI);

[0573] (Formula XVII); wherein: m is 2; n is an integer such that a PEG group having the structure of — (OCH2CH2)n— OCH3 has a molecular weight of about 30 kDa; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 124 that are not replaced.

[0574] 9. The IL-2 conjugate of embodiment 8, which is a pharmaceutically acceptable salt, solvate, or hydrate.

[0575] 10. A pharmaceutical composition comprising the IL-2 conjugate of any one of embodiments 1-9 and a pharmaceutically acceptable excipient.

[0576] I L A pharmaceutical composition comprising a mixture of IL-2 conjugates of embodiment 8 and a pharmaceutically acceptable excipient, wherein the mixture comprises IL-2 conjugates in which the [AzK_Ll_PEG30 kD] is an L-amino acid having the structure of Formula (XVI) and IL-2 conjugates in which the [AzK_Ll_PEG30 kD] is an L-amino acid having the structure of F ormul a (XVII) .

[0577] 12. An IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 85, wherein at least one amino acid at position K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, or L71 is replaced by the structure of Formula (I):

[0578] (Formula I) wherein:

[0579] Z is CH2 and Y is

[0580]

[0581] Z is CH2 and Y is or

[0582] Y is CH2 and Z is

[0583] W is a PEG group having a molecular weight of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa;

[0584] X has the structure:

[0585] X-l indicates the point of attachment to the preceding amino acid residue; and X+l indicates the point of attachment to the following amino acid residue. 13. The IL-2 conjugate of embodiment 12, which is a pharmaceutically acceptable salt, solvate, or hydrate.

[0586] 14. The IL-2 conjugate of embodiment 12, wherein the PEG group has a molecular weight of about 30 kDa.

[0587] 15. The IL-2 conjugate of embodiment 12, wherein the amino acid at position P64 is replaced by the structure of Formula (I).

[0588] 16. The IL-2 conjugate of embodiment 15, wherein the PEG group has a molecular weight of about 30 kDa.

[0589] 17. The IL-2 conjugate of embodiment 12, wherein the structure of Formula (I) has the structure of Formula (IV) or Formula (V):

[0590] (Formula V); wherein: W is a PEG group having a molecular weight of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa.

[0591] 18. The IL-2 conjugate of embodiment 17, wherein W is a PEG group having a molecular weight of about 30 kDa.

[0592] 19. A pharmaceutical composition comprising the IL-2 conjugate of embodiment 12 and a pharmaceutically acceptable excipient.

[0593] 20. A pharmaceutical composition comprising a mixture of IL-2 conjugates of embodiment 17 and a pharmaceutically acceptable excipient, wherein the mixture comprises IL-2 conjugates in which the structure of Formula (I) has the structure of Formula (IV) and IL-2 conjugates in which the structure of Formula (I) has the structure of Formula (V).

[0594] 21. A pharmaceutical composition comprising a mixture of IL-2 conjugates of embodiment 18 and a pharmaceutically acceptable excipient, wherein the mixture comprises IL-2 conjugates in which the structure of Formula (I) has the structure of Formula (IV) and IL-2 conjugates in which the structure of Formula (I) has the structure of Formula (V).

[0595] 22. The IL-2 conjugate of embodiment 12, wherein amino acid P64 is replaced by the structure of Formula (XII) or (XIII):

[0596] (Formula XII);

[0597]

[0598] (Formula XIII); wherein: n is an integer such that — (OCEECEEjn — OCH3 has a molecular weight of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 85 that are not replaced.

[0599] 23. The IL-2 conjugate of embodiment 22, which is a pharmaceutically acceptable salt, solvate, or hydrate.

[0600] 24. A pharmaceutical composition comprising the IL-2 conjugate of embodiment 22 and a pharmaceutically acceptable excipient.

[0601] 25. A pharmaceutical composition comprising a mixture of IL-2 conjugates of embodiment 22 and a pharmaceutically acceptable excipient, wherein the mixture comprises IL-2 conjugates in which amino acid P64 is replaced by the structure of Formula (XII) and IL-2 conjugates in which amino acid P64 is replaced by the structure of Formula (XIII).

[0602] 26. An IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 85, wherein the amino acid at position P64 is replaced by the structure of Formula (I):

[0603]

[0604] W is a PEG group having a molecular weight of about 30 kDa; X has the structure:

[0605] X-l indicates the point of attachment to the preceding amino acid residue; and X+l indicates the point of attachment to the following amino acid residue.

[0606] 27. The IL-2 conjugate of embodiment 26, which is a pharmaceutically acceptable salt, solvate, or hydrate.

[0607] 28. The IL-2 conjugate of embodiment 1, wherein the PEG has a molecular weight of about 30 kDa.

[0608] 29. The IL-2 conjugate of embodiment 1, wherein the IL-2 comprises an amino acid sequence having about 95% sequence identity to SEQ ID NO: 84.

[0609] 30. The IL-2 conjugate of embodiment 29, wherein the IL-2 comprises an amino acid sequence having about 97% sequence identity to SEQ ID NO: 84.

[0610] 31. The IL-2 conjugate of embodiment 1, wherein the IL-2 comprises an amino acid sequence having about 99% sequence identity to SEQ ID NO: 124.

[0611] 32. The pharmaceutical composition of embodiment 24, wherein the average molecular weight of — (OCEECEEjn — OCH3 in the IL-2 conjugates in the pharmaceutical composition is about 30 kDa. 33. The pharmaceutical composition of embodiment 25, wherein the average molecular weight of — (OCH2CH2)n— OCH3 in the IL-2 conjugates in the pharmaceutical composition is about 30 kDa.

[0612] IL-2 polypeptides and conjugates thereof and method of making said IL-2 polypeptides and conjugates thereof are described in further detail in WO2019028419, WO2020163532, and WO2024137864, each of which is incorporated herein.

[0613] Combination Therapy for the Treatment of Cancer

[0614] The disclosure provides methods of treating cancer with the combination of binding proteins that bind NKp46 on NK cells and CD 123 on cancer cells (CD 123 NKCEs) and IL-2 polypeptides and IL-2 conjugates thereof. The combination of the CD123 NKCEs of the disclosure and the IL-2 polypeptides and IL-2 conjugates thereof of the disclosure synergistically enhance cancer cell cytotoxicity.

[0615] Thus, in one embodiment, the disclosure provides a method of treating cancer in a subject, comprising administering to the subject: i) a binding protein comprising a first and a second antigen binding domain (ABD), wherein the first ABD binds specifically to human CD 123 and the second ABD binds specifically to human NKp46; and ii) an interleukin-2 (IL- 2) polypeptide or variant thereof, thereby treating the cancer in the subject.

[0616] In another embodiment, the disclosure provides a method of treating cancer, the method comprising administering an interleukin-2 (IL-2) polypeptide or variant thereof or conjugate thereof to a subject who has cancer and who has received a binding protein as described herein such that the subject receives a cancer treatment comprising the binding protein and the IL-2 polypeptide or variant thereof or conjugate thereof.

[0617] In another embodiment, the disclosure provides a method of treating cancer, the method comprising administering a binding protein described herein to a subject who has cancer and who has received an interleukin-2 (IL-2) polypeptide or variant thereof or conjugate thereof such that the subject receives a cancer treatment comprising the binding protein and the IL-2 polypeptide or variant thereof or conjugate thereof.

[0618] In another embodiment, the disclosure provides a method of inducing cytotoxicity of cancer cells, comprising contacting the cancer cells with: i) a binding protein comprising a first and a second antigen binding domain (ABD), wherein the first ABD binds specifically to human CD 123 and the second ABD binds specifically to human NKp46; and ii) an interleukin- 2 (IL-2) polypeptide, thereby inducing cytotoxicity of the cancer cells. In some embodiments, the cancer cells express CD 123.

[0619] In another embodiment, the disclosure provides for the use of a composition comprising a binding protein as described herein comprising a first and a second antigen binding domain (ABD), wherein the first ABD binds specifically to human CD 123 and the second ABD binds specifically to human NKp46; wherein the binding protein is administered in combination with an interleukin-2 (IL-2) polypeptide or variant thereof or conjugate thereof as described herein, in the treatment of cancer.

[0620] In another embodiment, the disclosure provides for the use of a composition comprising an interleukin-2 (IL-2) polypeptide or variant thereof or conjugate thereof as described herein; wherein the IL-2 polypeptide or variant thereof or conjugate thereof is administered in combination with a binding protein as described herein comprising a first and a second antigen binding domain (ABD), wherein the first ABD binds specifically to human CD 123 and the second ABD binds specifically to human NKp46, in the treatment of cancer.

[0621] In another embodiment, the disclosure provides for the use of i) a binding protein as described herein comprising a first and a second antigen binding domain (ABD), wherein the first ABD binds specifically to human CD 123 and the second ABD binds specifically to human NKp46; and ii) an interleukin-2 (IL-2) polypeptide or variant thereof or conjugate thereof as described herein, in the treatment of cancer.

[0622] In another embodiment, the disclosure provides for a composition comprising i) a binding protein as described herein comprising a first and a second antigen binding domain (ABD), wherein the first ABD binds specifically to human CD 123 and the second ABD binds specifically to human NKp46; and ii) an interleukin-2 (IL-2) polypeptide or variant thereof or conjugate thereof as described herein, for use in the treatment of cancer.

[0623] In another embodiment, the disclosure provides for: i) a binding protein as described herein comprising a first and a second antigen binding domain (ABD), wherein the first ABD binds specifically to human CD 123 and the second ABD binds specifically to human NKp46; and ii) an interleukin-2 (IL-2) polypeptide or variant thereof or conjugate thereof as described herein, for use in the treatment of cancer.

[0624] In another embodiment, the disclosure provides for the use of i) a binding protein as described herein comprising a first and a second antigen binding domain (ABD), wherein the first ABD binds specifically to human CD 123 and the second ABD binds specifically to human NKp46; and ii) an interleukin-2 (IL-2) polypeptide or variant thereof or conjugate thereof as described herein, for the manufacture of medicament for the treatment of cancer.

[0625] The terms “in combination with,” “administered in combination,” “administering a combination of,” and the like, as used in reference to the administration of multiple agents to a single subject refers to the administration of a first agent (e.g., an anti-CD123 x NKp46 binding protein) and a second agent (e.g., an interleukin-2 (IL-2) polypeptide or variant thereof or conjugate thereof) as part of a therapeutic regimen. Agents administered in combination are optionally further administered with an additional (i.e., third, fourth, fifth, etc.) supplementary agent or therapeutic treatment or regimen. Agents administered in combination may be administered simultaneously, contemporaneously, or sequentially. Agents administered in combination may be formulated and administered separately or may be co-formulated in single pharmaceutically acceptable formulation and the co-formulation is administered to a subject. A first agent is considered to be administered in combination with a second agent if the first and second agents are administered simultaneously, contemporaneously, or sequentially. A first agent is administered “simultaneously” with a second agent if first and second agents are administered within about 2 hours of each other. A first agent is administered “contemporaneously” with a second agent if first and second agents are administered within about 24 hours of each another.

[0626] When administered sequentially, administration of the first agent may provide a therapeutic effect over an extended time and the administration of the second agent administered while the therapeutic effect of the first agent persists in the subject such that the second agent is considered to be administered in combination with the first agent, even though the first agent may have been administered at a point in time significantly distant (e.g., days or weeks) from the time of administration of the second agent. In some embodiments, the biological effect of the administration of a first therapeutic agent may persist for a period of time beyond the point where there is a detectable level of the first therapeutic agent in the subject. In certain instances, a first agent and a second agent(s) are administered sequentially, e.g., where one agent is administered to the subject at least 24 hours prior to the administration of the second agent. When a first agent and second agent are administered to a subject sequentially, the first agent is considered to be administered “in combination with” with the second agent if at least 10%, alternatively at least 20%, alternatively at least 30%, alternatively at least 40%, alternatively at least 50%, alternatively at least 60%, alternatively at least 70%, alternatively at least 80%, alternatively at least 90%, alternatively at least 95% of the biological effect of the first agent persists in the subject at the time of administration of the second agent.

[0627] In some embodiments, the binding protein is administered to the subject prior to the IL- 2 polypeptide.

[0628] In some embodiments, the IL-2 polypeptide is administered to the subject prior to the binding protein.

[0629] In some embodiments, the binding protein and the IL-2 polypeptide thereof are administered contemporaneously (e.g., simultaneously) to the subject.

[0630] In some embodiments, the cancer is a CD 123 -expressing cancer.

[0631] In some embodiments, the cancer is a blood cancer.

[0632] In some embodiments, the cancer is myelodysplastic syndrome (MDS) or a lymphoproliferative disorder.

[0633] In some embodiments, the cancer is Acute Myeloid Leukemia (AML).

[0634] In some embodiments, cytotoxicity of cancer cells in the subject is induced after administration of the binding protein and the IL-2 polypeptide.

[0635] In some embodiments, NK cell activation in the subject is induced after administration of the binding protein and the IL-2 polypeptide.

[0636] In some embodiments, NK cell activation is determined by measuring CD69 expression from NK cells in the subject.

[0637] In some embodiments, administration of the binding protein and the IL-2 polypeptide to the subject results in increased release of one or more effector cytokines.

[0638] In some embodiments, the one or more effector cytokines are IFN-y, TNF-a, MIP-la, MIP-ip, Granzyme A, Granzyme B, and Lymphotactin.

[0639] In some embodiments, the binding protein described herein and the IL-2 polypeptide or variant thereof or conjugate thereof as described herein are in the same composition.

[0640] In some embodiments, the binding protein described herein and the IL-2 polypeptide or variant thereof or conjugate thereof as described herein are in separate compositions.

[0641] EMBODIMENTS OF THE DISCLOSURE

[0642] 1. A method of treating cancer in a subject, comprising administering to the subject: i) a binding protein comprising a first and a second antigen binding domain (ABD), wherein the first ABD binds specifically to human CD 123 and the second ABD binds specifically to human NKp46; and ii) an interleukin-2 (IL-2) polypeptide or variant thereof, thereby treating the cancer in the subj ect.

[0643] 2. The method of embodiment 1, wherein each of said ABD comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3); and wherein:

[0644] (i) the first ABD comprises:

[0645] - a VH1 comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 1 to 3 respectively or corresponding to the amino acid sequences of SEQ ID NO: 4 to 6 respectively, and

[0646] - a VL1 comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 7 to 9 respectively or corresponding to the amino acid sequences of SEQ ID NO: 10 to 12 respectively; and

[0647] (ii) the second ABD comprises:

[0648] - a VH2 comprising a CDR-H1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 13 to 15 respectively; the amino acid sequences of SEQ ID NO: 16 to 18 respectively; the amino acid sequences of SEQ ID NO: 19 to 21 respectively; the amino acid sequences of SEQ ID NO: 22 to 24 respectively; or the amino acid sequences of SEQ ID NO: 16, 25 and 26 respectively; and

[0649] - a VL2 comprising a CDR-L1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 27 to 29 respectively; the amino acid sequences of SEQ ID NO: 30 to 32 respectively; the amino acid sequences of SEQ ID NO: 33 to 35 respectively; the amino acid sequences of SEQ ID NO: 36 to 38 respectively; or the amino acid sequences SEQ ID NO: 39, 31 and 40 respectively. 3. The method of embodiment 1, wherein each of said ABD comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3); and wherein:

[0650] (i) the first ABD comprises:

[0651] - a VH1 comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 88 to 90 respectively, and

[0652] - a VL1 comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 94 to 96 respectively; and

[0653] (ii) the second ABD comprises:

[0654] - a VH2 comprising a CDR-H1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 13 to 15 respectively; the amino acid sequences of SEQ ID NO: 16 to 18 respectively; the amino acid sequences of SEQ ID NO: 19 to 21 respectively; the amino acid sequences of SEQ ID NO: 22 to 24 respectively; or the amino acid sequences of SEQ ID NO: 16, 25 and 26 respectively; and

[0655] - a VL2 comprising a CDR-L1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 27 to 29 respectively; the amino acid sequences of SEQ ID NO: 30 to 32 respectively; the amino acid sequences of SEQ ID NO: 33 to 35 respectively; the amino acid sequences of SEQ ID NO: 36 to 38 respectively; or the amino acid sequences SEQ ID NO: 39, 31 and 40 respectively.

[0656] 4. The method of embodiment 1, wherein each of said ABD comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3); and wherein:

[0657] (i) the first ABD comprises:

[0658] - a VH1 comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 91 to 93 respectively, and

[0659] - a VL1 comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 97 to 99 respectively; and (ii) the second ABD comprises:

[0660] - a VH2 comprising a CDR-H1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 13 to 15 respectively; the amino acid sequences of SEQ ID NO: 16 to 18 respectively; the amino acid sequences of SEQ ID NO: 19 to 21 respectively; the amino acid sequences of SEQ ID NO: 22 to 24 respectively; or the amino acid sequences of SEQ ID NO: 16, 25 and 26 respectively; and

[0661] - a VL2 comprising a CDR-L1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 27 to 29 respectively; the amino acid sequences of SEQ ID NO: 30 to 32 respectively; the amino acid sequences of SEQ ID NO: 33 to 35 respectively; the amino acid sequences of SEQ ID NO: 36 to 38 respectively; or the amino acid sequences SEQ ID NO: 39, 31 and 40 respectively.

[0662] 5. The method of any one of embodiments 1-4, wherein the binding protein comprises all or part of an immunoglobulin Fc region or variant thereof.

[0663] 6. The method of embodiment 5, wherein all or part of the immunoglobulin Fc region or variant thereof binds to a human Fc-y receptor.

[0664] 7. The method of any one of embodiments 1-6, wherein the binding protein comprises three polypeptide chains (I), (II) and (III) that form two ABDs, as defined below:

[0665] (I): light chain variable domain 1 A (VIA) - light chain constant domain 1 A (CIA) -Hingei

[0666] - (CH2-CH3)A;

[0667] (II): heavy chain variable domain IB (VIB) - heavy chain constant domain IB (CIB) - Hinge? - (CH2-CH3)B - LI - heavy chain variable domain 2A (V?A) - heavy chain constant domain 2 A (C2A) - Hinges; and

[0668] (III): light chain variable domain 2B (V2B) - light chain constant domain 2B (C2B), wherein:

[0669] VIA and VIB form a binding pair Vi (VHI / VLI);

[0670] V2A and V2B form a binding pair V2 (VH2 / VL2); CIA and CIB form a pair Ci (CH1 / CL) and C2A and C2B form a pair C2 (CH1 / CL) wherein CHI is an immunoglobulin heavy chain constant domain 1 and CL is an immunoglobulin light chain constant domain;

[0671] Hingei, Hinge2 and Hinges are identical or different and correspond to all or part of an immunoglobulin hinge region;

[0672] (Cu2-CH3)Aand (CH2-CH3)B are identical or different, and comprise an immunoglobulin heavy chain constant domain 2 (CH2) and an immunoglobulin heavy chain constant domain 3 (CH3);

[0673] LI is an amino acid linker.

[0674] 8. The method of embodiment 7, wherein:

[0675] CIB is an immunoglobulin heavy chain constant domain 1 (CHI);

[0676] C2A is an immunoglobulin heavy chain constant domain 1 (CHI);

[0677] CL corresponds to an immunoglobulin kappa light chain constant domain (CK); (CH2-CH3)A corresponds to the amino acid sequence of SEQ ID NO: 69;

[0678] (CH2-CH3)B corresponds to the amino acid sequence of SEQ ID NO: 70;

[0679] Hingei corresponds to the amino acid sequence of SEQ ID NO: 74;

[0680] Hinge2 corresponds to the amino acid sequence of SEQ ID NO: 75;

[0681] Hinges corresponds to the amino acid sequence of SEQ ID NO: 77;

[0682] Li corresponds to the amino acid sequence of SEQ ID NO: 76.

[0683] 9. The method of any one of embodiments 5-8, wherein residue N297 of the Fc region or variant thereof according to EU numbering comprises a N-linked glycosylation.

[0684] 10. The method of any one of embodiments 5-9, wherein the all or part of the Fc region or variant thereof binds to a human CD16A (FcyRIII) polypeptide.

[0685] 11 The method of any one of embodiments 5-10, wherein the Fc region or variant thereof is of an IgGl, IgG2, IgG3, or IgG4 isotype.

[0686] 12. The method of any one of embodiments 1-11, wherein the binding protein comprises at least two polypeptide chains linked by at least one disulfide bridge. 13. The method of any one of embodiments 7-12, wherein the polypeptide chains (I) and (II) are linked by at least one disulfide bridge between CIA and Hinge? and / or wherein the polypeptide chains (II) and (III) are linked by at least one disulfide bridge between Hinges and C?B.

[0687] 14. The method of any one of embodiments 7-12, wherein VIA is VLI and VIB is VHI.

[0688] 15. The method of any one of embodiments 7-12, wherein V2A is VH2 and V?B is VL2.

[0689] 16. The method of any one of embodiments 2-15, wherein:

[0690] (a) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 27; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 28; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29;

[0691] (b) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32; (c) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 34; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35;

[0692] (d) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 37; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38;

[0693] (e) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 39; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40; (f) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 13; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 27; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 28; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29;

[0694] (g) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32;

[0695] (h) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 19; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35; (i) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 22; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38; or

[0696] (j) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 39; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0697] 17. The method of any one of embodiments 2-16, wherein:

[0698] (a) VHI and VLI corresponds to the amino acid sequences of SEQ ID NO: 41 and 43 respectively or corresponds to the amino acid sequences of SEQ ID NO: 42 and 44 respectively; and / or

[0699] (b) VH2 and VL2 corresponds 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; the amino acid sequences of SEQ ID NO: 51 and 59 respectively; or the amino acid sequences of SEQ ID NO: 52 and 60 respectively.

[0700] 18. The method of embodiment 17, wherein:

[0701] (a) VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 45; VL2 comprises the amino acid sequence of SEQ ID NO: 53;

[0702] (b)Vm comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 46; VL2 comprises the amino acid sequence of SEQ ID NO: 54;

[0703] (C)VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 47; VL2 comprises the amino acid sequence of SEQ ID NO: 55;

[0704] (d)Vm comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 48; VL2 comprises the amino acid sequence of SEQ ID NO: 56;

[0705] (e)Vm comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 49; VL2 comprises the amino acid sequence of SEQ ID NO: 57;

[0706] (f) VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 50; VL2 comprises the amino acid sequence of SEQ ID NO: 58;

[0707] (g)Vm comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 51; VL2 comprises the amino acid sequence of SEQ ID NO: 59;

[0708] (h)Vm comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 52; VL2 comprises the amino acid sequence of SEQ ID NO: 60;

[0709] (i) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 45; VL2 comprises the amino acid sequence of SEQ ID NO: 53; (j) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 46; VL2 comprises the amino acid sequence of SEQ ID NO: 54;

[0710] (k) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 47; VL2 comprises the amino acid sequence of SEQ ID NO: 55;

[0711] (l) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 48; VL2 comprises the amino acid sequence of SEQ ID NO: 56;

[0712] (m) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 49; VL2 comprises the amino acid sequence of SEQ ID NO: 57;

[0713] (n) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 50; VL2 comprises the amino acid sequence of SEQ ID NO: 58;

[0714] (o) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 51; VL2 comprises the amino acid sequence of SEQ ID NO: 59;

[0715] (p) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 52; VL2 comprises the amino acid sequence of SEQ ID NO: 60.

[0716] 19. The method of any one of embodiments 7-18, wherein:

[0717] - polypeptide (I) comprises an amino acid sequence of SEQ ID NO: 64;

[0718] - polypeptide (II) comprises an amino acid sequence of SEQ ID NO: 65; and

[0719] - polypeptide (III) comprises an amino acid sequence of SEQ ID NO: 66.

[0720] 20. The method of any one of embodiments 7-19, wherein:

[0721] - polypeptide (I) consists of an amino acid sequence of SEQ ID NO: 64;

[0722] - polypeptide (II) consists of an amino acid sequence of SEQ ID NO: 65; and

[0723] - polypeptide (III) consists of an amino acid sequence of SEQ ID NO: 66. 21. The method of any one of embodiments 1-20, wherein the IL-2 polypeptide comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 84.

[0724] 22. The method of any one of embodiments 1-20, wherein the IL-2 polypeptide comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 85.

[0725] 23. The method of any one of embodiments 1-22, wherein the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 85 in which at least one amino acid residue is replaced by an unnatural amino acid.

[0726] 24. The method of embodiment 23, wherein the position of the at least one unnatural amino acid is selected from K34, T36, R37, T40, F41, K42, F43, Y44, E59, E60, E61, K63, P64, E67, V68, N70, L71, M103, C104, and Y107, wherein the residue positions correspond to the positions 34, 36, 37, 40, 41, 42, 43, 44, 60, 61, 63, 64, 67, 68, 70, 71, 103, 104, and 106 as set forth in SEQ ID NO: 85.

[0727] 25. The method of any one of embodiments 23-24, wherein the position of the at least one unnatural amino acid is selected from T36, R37, T40, F41, K42, F43, Y44, E60, E61, P64, E67, and L71 , wherein the residue positions correspond to the positions 36, 37, 40, 41, 42, 43, 44, 60, 61, 64, 67, and 71 as set forth in SEQ ID NO: 85.

[0728] 26. The method of any one of embodiments 23-24, wherein the position of the at least one unnatural amino acid is selected from K34, K63, V68, N70, M103, C104, and Y106, wherein the residue positions correspond to the positions 34, 63, 68, 70, 103, 104, and 106 as set forth in SEQ ID NO: 85.

[0729] 27. The method of any one of embodiments 23-24, wherein the position of the at least one unnatural amino acid is selected from T36, R37, T40, Y44, E60, E67, and L71, wherein the residue positions correspond to the positions 36, 37, 40, 44, 60, 67, and 71 as set forth in SEQ ID NO: 85. 28. The method of any one of embodiments 23-24, wherein the position of the at least one unnatural amino acid is selected from F41, K42, F43, E61, and P64, wherein the residue positions correspond to the positions 41, 42, 43, 61, and 64 as set forth in SEQ ID NO: 85.

[0730] 29. The method of any one of embodiments 23-28, wherein the at least one unnatural amino acid:

[0731] • is a lysine analogue;

[0732] • comprises an aromatic side chain;

[0733] • comprises an azido group;

[0734] • comprises an alkyne group; or

[0735] • comprises an aldehyde or ketone group.

[0736] 30. The method of any one of embodiments 23-28, wherein the at least one unnatural amino acid does not comprise an aromatic side chain.

[0737] 31. The method of any one of embodiments 23-30, wherein the at least one unnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN- L-lysine, norbomene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine, 2- amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p- acetyl-L-phenylalanine, p- azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2- amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3- methyl-phenylalanine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p- azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p- bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, 0-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O- acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2- amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3- (phenylselanyl)propanoic, or selenocysteine.

[0738] 32. The method of any one of embodiments 23-31, wherein the IL-2 polypeptide is covalently attached to a conjugating moiety through the at least one unnatural amino acid. 33. The method of embodiment 32, wherein the conjugating moiety comprises a water- soluble polymer, a lipid, a protein, or a peptide.

[0739] 34. The method of embodiment 32, wherein the water-soluble polymer comprises polyethylene glycol (PEG), polypropylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N- acryloylmorpholine), or a combination thereof.

[0740] 35. The method of embodiment 32, wherein the water-soluble polymer comprises a PEG molecule.

[0741] 36. The method of embodiment 34 or 35, wherein the PEG molecule is a linear PEG.

[0742] 37. The method of embodiment 34 or 35, wherein the PEG molecule is a branched PEG.

[0743] 38. The method of embodiment 32, wherein the water-soluble polymer comprises a polysaccharide.

[0744] 39. The method of embodiment 38, wherein the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl- starch (HES).

[0745] 40. The method of embodiment 33, wherein the lipid comprises a fatty acid.

[0746] 41. The method of embodiment 40, wherein the fatty acid comprises from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms. 42. The method of embodiment 40 or 41, wherein the fatty acid is a saturated fatty acid.

[0747] 43. The method of embodiment 33, wherein the protein comprises an albumin, a transferrin, or a transthyretin.

[0748] 44. The method of embodiment 32, wherein the conjugating moiety comprises a toll-like receptor (TLR) agonist.

[0749] 45. The method of any one of embodiments 32-44, wherein the conjugating moiety is indirectly bound to the at least one unnatural amino acid of the IL-2 polypeptide through a linker.

[0750] 46. The method of embodiment 45, wherein the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or a non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof.

[0751] 47. The method of any one of embodiments 19-46, wherein the IL-2 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof.

[0752] 48. The method of embodiment 47, wherein the N-terminal deletion comprises a deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from the N-terminus, wherein the residue positions are in reference to the positions in SEQ ID NO: 85.

[0753] 49. The method of embodiment 47, wherein the C-terminal deletion comprises a deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues from the C-terminus, wherein the residue positions are in reference to the positions in SEQ ID NO: 85.

[0754] 50. The method of any one of embodiments 32-49, wherein the conjugating moiety or the unnatural amino acid impairs or blocks the binding of IL-2 with IL-2Ra. 51. The method of any one of embodiments 1-20, wherein the IL-2 polypeptide comprises an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 85, wherein the amino acid at position P64 is replaced by the structure of Formula (I):

[0755] W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; q is 1, 2, or 3;

[0756] X has the structure: wherein X-l indicates the point of attachment to the preceding amino acid residue; and wherein X+l indicates the point of attachment to the following amino acid residue.

[0757] 52. The method of embodiment 51 , wherein the PEG group has an average molecular weight of about 30 kDa.

[0758] 53. The method of embodiment 51, wherein Z is CH2 and Y is

[0759] 55. The method of embodiment 51, wherein Z is CH2 and Y is

[0760] 57. The method of embodiment 51 or 52, wherein the IL-2 conjugate comprises (i) an IL-2 conjugate in which the structure of Formula (I) has the structure of Formula (IV), and / or (ii) an IL-2 conjugate in which the structure of Formula (I) has the structure of or Formula (V):

[0761] Formula (V); wherein: q is 1, 2, or 3;

[0762] X has the structure: wherein X-l indicates the point of attachment to the preceding amino acid residue; and wherein X+l indicates the point of attachment to the following amino acid residue.

[0763] 58. The method of embodiment 51 or 52, wherein the IL-2 conjugate comprises (i) an IL-2 conjugate in which the structure of Formula (I) has the structure of Formula (XII), and / or (ii) an IL-2 conjugate in which the structure of Formula (I) has the structure of or Formula (XIII):

[0764] Formula (XII);

[0765] Formula (XIII); wherein: n is an integer such that a PEG group having the structure of -(OCEECEEjn-OCEE has a molecular weight of about 30 kDa; q is 1, 2, or 3; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 85 that are not replaced.

[0766] 59. The method of any one of embodiments 51-58, wherein q is 1.

[0767] 60. The method of any one of embodiments 51-58, wherein q is 2.

[0768] 61. The method of any one of embodiments 51-58, wherein q is 3.

[0769] 62. The method of any one of embodiments 31-61, wherein the IL-2 polypeptide that is covalently attached to a conjugating moiety has reduced binding between the IL-2 polypeptide and interleukin 2 receptor alpha (IL-2Ra) but retains significant binding with interleukin 2 Py receptor (IL-2R Py) signaling complex to form an IL-2 / IL- 2RPy complex, wherein the reduced binding to IL-2Ra is compared to binding between a wild-type IL-2 polypeptide and IL-2Ra.

[0770] 63. The method of embodiment 62, wherein the reduced binding is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% reduced binding to IL-2Ra relative to a wild-type IL-2 polypeptide.

[0771] 64. The method of embodiment 62, wherein the reduced binding is about 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more reduced binding to IL-2Ra relative to a wild-type IL-2 polypeptide. 65. The method of any one of embodiments 1-64, wherein the binding protein is administered to the subject prior to the IL-2 polypeptide.

[0772] 66. The method of any one of embodiments 1-64, wherein the IL-2 polypeptide is administered to the subject prior to the binding protein.

[0773] 67. The method of any one of embodiments 1-64, wherein the binding protein and the IL-2 polypeptide thereof are administered contemporaneously to the subject.

[0774] 67. The method of any one of embodiments 1-64, wherein the cancer is a blood cancer.

[0775] 68. The method of any one of embodiments 1-64, wherein the cancer is myelodysplastic syndrome (MDS) or a lymphoproliferative disorder.

[0776] 69. The method of any one of embodiments 1-64, wherein the cancer is Acute Myeloid Leukemia (AML).

[0777] 70. The method of any one of embodiments 1-69, wherein cytotoxicity of cancer cells in the subject is induced after administration of the binding protein and the IL-2 polypeptide.

[0778] 71. The method of any one of embodiments 1-70, wherein NK cell activation in the subject is induced after administration of the binding protein and the IL-2 polypeptide.

[0779] 72. The method of 71, wherein NK cell activation is determined by measuring CD69 expression from NK cells in the subject.

[0780] 73. The method of any one of embodiments 1-72, wherein administration of the binding protein and the IL-2 polypeptide to the subject results in increased release of one or more effector cytokines.

[0781] 74. The method of embodiment 73, wherein the one or more effector cytokines are IFN- y, TNF-a, MIP-la, MIP-ip, Granzyme A, Granzyme B, and Lymphotactin. SEQUENCE LISTING

[0782] In the protein sequences notation used herein, the left-hand direction is the amino terminal direction (the “N terminus” or “N-term”) and the right-hand direction is the carboxyl-terminal direction (the “C terminus” or “C-term”), in accordance with standard usage and convention.

[0783]

[0784] EXAMPLES

[0785] EXAMPLE 1 - Cytotoxicity of CD123 NKCE in combination with an exemplary IL-2 conjugate.

[0786] The CD123 NKCE described in the examples is a humanized multi-specific Natural Killer cell engager (NKCE) that binds to the human CD 123 tumor-associated antigen with one arm and to the activating NK-cell receptor, NKp46, with the other. CD123 NKCE also possesses a competent Fc domain that is able to promote antibody-dependent cell-mediated cytotoxicity (ADCC) via the activating receptor CD 16 (FcyRIIIa) expressed on NK cells. Coengagement of NK and tumor cells by the construct leads to the formation of an immunological synapse between NK and tumor cells, which induces NK cell activation followed by degranulation with release of cytotoxic granules like perforin and granzymes, resulting in CD 123 -positive tumor cell killing (FIG. 3). CD123 is over-expressed in Acute Myeloid Leukemia (AML).

[0787] An exemplary IL-2 conjugate for use in combination with a CD123 NKCE is pegenzileukin, which is a PEGylated, engineered variant of interleukin 2 (IL-2) comprising an unnatural amino acid for use in site-specific pegylation, where a PEG chain is attached to the unnatural amino acid. Such an exemplary IL-2 conjugate is configured to prevent the binding of the IL-2 moiety to IL-2 alpha-receptor (IL-2R-alpha), thereby blocking the IL-2Ralpha- mediated activation of CD4-positive regulatory and / or immunosuppressive T cells (e.g., Tregs). Such an exemplary IL-2 conjugate retains near-native binding to the IL-2 beta-gamma receptors that promote NK cell activation and proliferation without the alpha-mediated immunosuppressive effects of regulatory T cells or eosinophil-mediated vascular leak syndrome.

[0788] In the present Example, the cytotoxic effect of the CD 123 NKCE in combination with pegenzileukin and in presence of healthy donor-derived NK cells (HD NK cells) against AML cell lines (THP-1 and MOLM-13) was evaluated in vitro.

[0789] Materials and Methods

[0790] Cytotoxicity assay by Incucyte®

[0791] Cytotoxicity was evaluated using the Incucyte® live-cells analysis system which allows to quantify the number of live fluorescent target cells over time. First, the CD123 NKCE (at 1, 10 or 100 ng / mL) or its isotype control (7G3-N31 S-F6-Icb for cytotoxicity experiments against THP-1 GFP cells and IC-F25-3D9H1L1 for cytotoxicity experiments against MOLM-13 RFP cells; also tested at 1, 10 or 100 ng / mL) were distributed in the appropriate wells of an F-bottom (flat-bottom) 96-well plate coated with poly-D-lysine. 7G3-N31S is a CD123 monoclonal antibody and 3D9H1L1 is an NKp46 monoclonal antibody). Next, pegenzileukin (at 100 ng / mL) was added into the appropriate wells to be combined with the CD123 NKCE (or the appropriate isotype control).

[0792] Then, MOLM-13 RFP or THP-1 GFP target cells (T) and resting effector HD NK cells (E) were successively added in each well to obtain E:T ratios of 1 : 1 (20000 E and 20000 T tumor cells) and 1 :2 (10000 E and 20000 T tumor cells; only for MOLM-13). Each condition was performed at least in duplicate. It has to be noted that: 1) the CD123 NKCE or its isotype control were tested only at 1 and 10 ng / mL in one experiment (Assay number PP29, E:T ratio of 1 : 1 against THP-1 cells); 2) the dose of Ing / ml was not evaluated for the E:T ratio of 1 :2 against MOLM- 13.

[0793] The growth / proliferation of tumor cells was monitored by cell imaging up to 72 hours using the Incucyte® S3 with standard scans every 2 hours for THP-1 and every 4 hours for MOLM-13. The IncucyteS3 software was used to analyze images. Tumor cells growth / proliferation was calculated by counting the number of cells at each time point normalized to the number of cells at time zero and expressed as a percentage.

[0794] For all combination studies, suboptimal concentrations of the CD123 NKCE (up to 100 ng / mL) and low E:T ratios (1 : 1 or 1 :2) were used to ensure observing a potential additive effect of pegenzileukin. The dose of 100 ng / mL of pegenzileukin used in these studies corresponds to the EC90 obtained in pSTAT5 and NK cell proliferation assay in vitro. This dose also correlates with the plasma concentration in subjects treated with pegenzileukin at a dose of 24 Pg / kg- AML cell lines

[0795] The human AML cells MOLM-13 RFP and THP-1 GFP were used in the study. These cells were used as they express high levels of CD 123.

[0796] MOLM-13 RFP

[0797] MOLM-13 RFP were generated by infecting MOLM-13 (DSMZ, ACC: 554) with the Incucyte® Nuclight Red Lentivirus (Sartorius #4476) to express a nuclear-restricted mKate2 (red fluorescent protein, RFP). MOLM-13 RFP were cultured in RPMH640 (Gibco #31870- 025) supplemented with 20 % foetal bovine serum (FBS) (Eurobio #CVFSVF06-01) and 1% L-Glutamine (Gibco #25030-081).

[0798] THP-1 GFP THP-1 GFP were generated by infecting THP-1 (ATCC, TIB-202™) with the Incucyte® Nuclight Green Lentivirus (Sartorius #4475) to express Green Fluorescent Protein (GFP). THP-1 GFP were cultured in RPMI1640 (Gibco #31870-025) supplemented with 10 % FBS (Eurobio #CVFSVF06-01) and 1% L-Glutamine (Gibco #25030-081).

[0799] Human NK cells

[0800] Human peripheral blood mononuclear cells (PBMCs) isolation

[0801] Buffy coats from healthy donors (HD) were supplied by EFS He de France (Rungis site). Immediately after reception, the qualified buffy coats were put under gentle agitation overnight at room temperature. The following day, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. The entire blood bag was collected and diluted with 60 mL of sterile Dulbecco’s phosphate-buffered saline (DPBS). Fifteen mL of Ficoll-Paque™ Plus (Cytiva #17-1440-02) were put into the center of four sepMate™-50 tubes (Stemcell Technologies #85450). Then, approximately 25 mL of the diluted blood were added into the sepMate™-50 tubes containing Ficoll-Paque™ Plus solution, on the edges of the tube, gently. The tubes were centrifuged at 1200g for 20 minutes at room temperature. The PBMCs were recovered in a 50 mL tube. Then, DPBS was added to have a final volume of 50 mL. The tube was centrifuged at 400g for 10 min at room temperature. The supernatant was removed, and the pellet was resuspended in 30 mL of RPMH640 medium (Gibco #31870-025) supplemented with 10 % FBS (Eurobio #CVFSVF06-01) and 1% L- Glutamine (Gibco #25030-081).

[0802] NK cell isolation

[0803] Human NK cells were purified from PBMCs with the MACSXpress Whole Blood NK cell isolation kit from Miltenyi Biotec (#130-127-695) by negative selection using manual magnetic labelling and the MACSxpress separator (Miltenyi Biotec #130-098-308). The purification was performed according to the protocol recommended by the supplier for 30 mL of whole blood. Once purified, NK cells were cultured in RPMH640 medium supplemented with 10 % FBS (Eurobio #CVFSVF06-01) and 1% L-Glutamine (Gibco #25030-081). The cells were left to rest overnight at 37°C with 5% CO2 before being used.

[0804] Cytotoxicity assay by Incucyte®: objective of the analysis The statistical analysis corresponds to the objective of comparison of each single compound to the CD 123 NKCE combined with pegenzileukin.

[0805] For each replicate, the Area Under the Curve (AUC) was computed from 0 hours to 72 hours according to the following formula:

[0806] Where: h is hour increasing in steps of 2 for THP-1 and 4 for MOLM-13; h_tis the previous measured timepoint;

[0807] Count is the number of normalized THP — 1 / MOLM — 13 cells measured Methodology applied

[0808] For each tumor cell line and each E:T ratio, areas under the curves were summarized by compound using descriptive statistics (Mean, Standard deviation (SD), Median, First (QI) and Third (Q3) Quartile). Statistical analysis was performed independently for THP-1 and each E:T ratio of MOLM-13. For each cell line and each E:T ratio, a mixed model was performed on AUC with fixed factors Group and Dose and random factors Experiment, Donor, Donor* Group.

[0809] Nevertheless, for E:T ratio=l :2 of MOLM-13, since only one donor was measured on one of the two experiments, it was not possible to include Experiment effect due to a lack of information. Hence, random factors were only donor and donor*group.

[0810] For E:T ratio=l : l of MOLM-13, based on inspection of residuals from the model, heterogeneous variance between groups was specified for Isotype control (IC)+pegenzileukin over all other groups.

[0811] A Dunnett’s adjustment on pairwise comparisons to the CD 123 NKCE combined with pegenzileukin was performed by dose to correct p-values for multiplicity. The statistical analyses were performed using SAS 9.4 for Windows 10. R version 4.1.2 was used for graphical representations. A probability less than 5% (p<0.05) was considered as significant.

[0812] Example 1 - Results

[0813] In vitro cytotoxic activity against THP-1 cells over time in presence of HD NK cells

[0814] The in vitro cytotoxic activity of the CD 123 NKCE in combination with pegenzileukin against THP-1 GFP cells was assessed overtime (up to 72 hours) by Incucyte®. the CD123 NKCE or its isotype control (IC: 7G3-N31 S-F6-Icb, which does not bind to NKp46 and is Fc- silent) were tested at two different concentrations (1 and 10 ng / mL); HD NK cells from 4 donors were tested at E:T ratio of 1 : 1. All data related to the AUC for each compound at each dose are presented in Table 1A. the CD123 NKCE induced cytotoxicity of THP-1 cells (in presence of HD NK cells) at either 1 ng / mL or 10 ng / mL, with the CD123 NKCE 10 ng / mL showing more potency than the CD 123 NKCE 1 ng / mL (FIG. 4A-FIG. 4B and FIG. 5).

[0815] Likewise, pegenzileukin induced cytotoxicity of THP-1 cells (in presence of HD NK cells) at 100 ng / mL (in presence of IC at both 1 ng / ml and 10 ng / mL - FIG. 4A-FIG. 4B). For each dose, all single compounds were significantly different from combination the CD 123 NKCE + pegenzileukin, (Table IB and FIG. 4A-FIG. 4B). Table 1A - THP-1 - Descriptive table of AUC for each compound at each dose of the CD123

[0816] NKCE. Pegenzileukin used atlOO ng / mL.

[0817] Abbreviations: SD: Standard deviation; QI: first quartile; Q3: third quartile Table IB - THP-1 - Comparison of each single compound to combination of the CD123 NKCE and pegenzileukin. Pegenzileukin used atlOO ng / mL. In vitro cytotoxic activity against MOLM-13 cells over time in presence of HD NK cells

[0818] The in vitro cytotoxic activity of the CD 123 NKCE in combination with pegenzileukin against MOLM-13 RFP was assessed overtime (up to 72h) by Incucyte®. The CD 123 NKCE or its isotype control (IC-F25-3D9H1L1) were tested at the concentration of 1, 10 or 100 ng / mL at the E:T ratio of 1 : 1 (with HD NK cells from 4 donors), and at the concentration of 10 or 100 ng / mL at the E:T ratio of 1 :2 (with HD NK cells from 3 donors). All data related to the AUC for each compound at each dose and both E:T ratios are presented in Table 1C.

[0819] Table 1C - MOLM-13 - Descriptive table of AUC for each compound at each dose of the CD123 NKCE. Pegenzileukin used atlOO ng / mL.

[0820] Abbreviations: SD: Standard deviation; QI: first quartile; Q3: third quartile

[0821] In vitro cytotoxic activity against MOLM-13 cells at E:T ratio 1 : 1 in presence of HD NK cells Cytotoxicity of MOLM-13 cells was induced by the CD123 NKCE (in presence of HD NK cells) at 1 ng / mL, 10 ng / mL or 100 ng / mL (FIG. 6A-FIG. 6C and FIG. 7). Cytotoxicity of MOLM-13 cells was also induced by pegenzileukin (in presence of HD NK cells) at 100 ng / mL in presence of IC (at all concentrations, FIG. 6A-FIG. 6C). For each dose, all single compounds were significantly different from combination the CD 123 NKCE + pegenzileukin, (Table ID and FIG. 6A-FIG. 6C and FIG. 7), with pegenzileukin at 100 ng / mL in combination with the CD123 NKCE at either 10 ng / mL or 100 ng / mL leading to complete killing of MOLM-13 cells (FIG. 6A-FIG. 6C).

[0822] Table ID - MOLM-13 E:T ratio=l : l - Comparison of each single compound to combination CD123 NKCE +pegenzileukin. Pegenzileukin used atlOO ng / mL.

[0823] In vitro cytotoxic activity against MOLM-13 cells at E:T ratio 1 :2 in presence of HD NK cells Cytotoxicity of MOLM-13 cells was induced by the CD123 NKCE (in presence of HD

[0824] NK cells) at 10 ng / mL and 100 ng / mL (FIG. 8A-8C). Cytotoxicity of MOLM-13 cells was also induced by pegenzileukin (in presence of HD NK cells) at 100 ng / mL in presence of IC (at both concentrations, FIG. 8A-8C). For each dose, all single compounds were significantly different from combination the CD123 NKCE + pegenzileukin. (Table IE and FIG. 8A-8C).

[0825] Table IE - MOLM-13 E:T ratio=l : l - Comparison of each single compound to combination CD123 NKCE+pegenzileukin. Pegenzileukin used atlOO ng / mL.

[0826] The results of Example 1 demonstrate that pegenzileukin combined with the CD 123 NKCE at different concentrations induced significantly superior cytotoxicity against AML cell models as compared to single agents, with NK cell-mediated killing at 1 : 1 and 1 :2 E:T ratios.

[0827] EXAMPLE 2 - In vitro effect of CD123 NKCE in combination with pegenzileukin on NK cell activation and cytokine / chemokine production

[0828] The aim of this study was to evaluate the effect of the CD123 NKCE in combination with pegenzileukin on NK cell activation and effector cytokine / chemokine release in presence of MOLM-13 AML tumor cells in vitro.

[0829] Example 2 - Materials and Methods

[0830] NK cell activation by flow cytometry the CD123 NKCE (at 10, 100 or 1000 ng / mL) and its isotype control (IC-F25- 3D9H1L1; at 100 or 1000 ng / ml) were distributed in the appropriate wells of an F-bottom 96- well plate. Next, pegenzileukin (at 100 ng / mL) was added to the appropriate wells to be combined with the CD123 NKCE (or added to isotype control). Then, 20000 MOLM-13 target cells (T) and 20000 resting effector HD NK cells (E) were added into each well to obtain a E:T ratio of 1 : 1. Each condition was performed in duplicate. The plates were spun down for 1 min at 100g then incubated at 37°C for 24 hours. After treatment, the plate' s content was transferred to U-bottom 96-well plates. Cells were centrifuged for 5 minutes at 300g, the supernatant removed and stored at -20°C for the experiment (see below). Cell pellets were washed once in 200 pL of autoMACS® Running Buffer (Miltenyi Biotec; #130-091-221).

[0831] Cell labelling was preceded by blocking the Fey receptors with the addition of human FcR blocking reagent (Miltenyi Biotec; #130-059-901) for 15 min at 4°C. Then, 100 pL of running buffer was added to wash the cells at 300g for 5 min. Cell labelling was performed for 20 min at 4°C in 100 pL of running buffer using APC mouse anti-human CD69 (clone FN50; Miltenyi Biotec; #130-113-520; 1 / 200) and PE mouse anti-human CD123 (clone 9F5; BD Biosciences; #555644; 1 / 200). Then, cells were washed twice with running buffer and resuspend in 80-100pL of D-PBS. To additionally assess cell viability, DAPI (Miltenyi Biotec, #130-111-570) was added in each well before plate reading by flow cytometry. Samples were then analyzed with the MACSQuant® Analyser 16 flow cytometer (Miltenyi Biotec). Data were analyzed with Flow Jo software using the following strategy: the NK cell population excluding debris was gated, followed by gating for single cells. From single cells, CD123 positive cells were excluded, and viable DAPI negative NK cells quantified. From DAPI negative cells, the expression of CD69 was quantified with APC fluorochrome and expressed as median of fluorescence intensity (MFI).

[0832] NK cell activation was determined by assessing the percentage of CD69 positive NK cells and the MFI of CD69 on NK cells by flow cytometry.

[0833] Effector cytokine / chemokine release by Mesoscale discovery (MSP)

[0834] The U-Plex Immuno-Oncology Group 1 (human) kit (#K151AEM-2 for INF-y, TNF- a, MIP-la (CCL3), MIP-ip (CCL4), Granzyme A and B) and R-PLEX® human Lymphotactin antibody set (#F21ACV-3 for Lymphotactin) were used to assess the release of effector cytokines and chemokines from NK cells after 24 and 48 hours of stimulation. Following manufacturer' s instruction, the multiplex coating solution was prepared by coupling a unique biotinylated capture antibody with a different linker to associate one spot per well to one capture antibody. Then, the U-plates were coated with the multiplex coating solution. Cell culture supernatants previously collected were thawed and added. In parallel, an 8-point calibration / standard curve with 4-fold serial dilution steps and a zero-calibrator blank was prepared. Then, the plates were incubated for 2 hours at room temperature with gentle shaking and then washed 3 times with 150 pL / well of washing buffer (0,05 % Tween in D-PBS).

[0835] After adding a solution of detection antibodies conjugated with electrochemiluminescent labels (MSD SULFO-TAG), the plates were incubated for 1 hour at room temperature with gentle shaking. Then, an MSD buffer was added to create an appropriate chemical environment for electrochemiluminescence (ECL) reaction. The plates were loaded into an MSD® QuickPlex instrument which detects excitation through the electrodes located in the bottom of each plate. Electricity was then applied to the plate electrodes by the MSD® QuickPlex instrument leading to light emission by SULFO-TAG labels (proportional to the quantity of analyte in the sample). Light intensity was then measured to quantify analytes in the sample. Analyte concentrations were determined from ECL signals by backfitting to the calibration curve established with a Four Parameter Logistic (4PL) regression. Data were analyzed with MSD Discovery Workbench® V4.

[0836] MOLM-13 cells (DSMZ, ACC: 554) were cultured in RPMI1640 (Gibco #31870-025) supplemented with 20 % foetal bovine serum (FBS) (Eurobio #CVFSV...

Claims

1. CLAIMS1. A method of treating cancer in a subject, comprising administering to the subject: i) a binding protein comprising a first and a second antigen binding domain (ABD), wherein the first ABD binds specifically to human CD 123 and the second ABD binds specifically to human NKp46; and ii) an interleukin-2 (IL-2) polypeptide or variant thereof, thereby treating the cancer in the subj ect.

2. The method of claim 1, wherein each of said ABD comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3); and wherein:(i) the first ABD comprises:- a VH1 comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 1 to 3 respectively or corresponding to the amino acid sequences of SEQ ID NO: 4 to 6 respectively, and- a VL1 comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 7 to 9 respectively or corresponding to the amino acid sequences of SEQ ID NO: 10 to 12 respectively; and(ii) the second ABD comprises:- a VH2 comprising a CDR-H1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 13 to 15 respectively; the amino acid sequences of SEQ ID NO: 16 to 18 respectively; the amino acid sequences of SEQ ID NO: 19 to 21 respectively; the amino acid sequences of SEQ ID NO: 22 to 24 respectively; or the amino acid sequences of SEQ ID NO: 16, 25 and 26 respectively; and- a VL2 comprising a CDR-L1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 27 to 29 respectively; the amino acid sequences of SEQ ID NO: 30 to 32 respectively; the amino acid sequences of SEQ ID NO: 33 to 35 respectively; the amino acid sequences of SEQ ID NO: 36 to 38 respectively; orthe amino acid sequences SEQ ID NO: 39, 31 and 40 respectively.

3. The method of claim 1, wherein each of said ABD comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3); and wherein:(i) the first ABD comprises:- a VH1 comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 88 to 90 respectively, and- a VL1 comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 94 to 96 respectively; and(ii) the second ABD comprises:- a VH2 comprising a CDR-H1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 13 to 15 respectively; the amino acid sequences of SEQ ID NO: 16 to 18 respectively; the amino acid sequences of SEQ ID NO: 19 to 21 respectively; the amino acid sequences of SEQ ID NO: 22 to 24 respectively; or the amino acid sequences of SEQ ID NO: 16, 25 and 26 respectively; and- a VL2 comprising a CDR-L1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 27 to 29 respectively; the amino acid sequences of SEQ ID NO: 30 to 32 respectively; the amino acid sequences of SEQ ID NO: 33 to 35 respectively; the amino acid sequences of SEQ ID NO: 36 to 38 respectively; or the amino acid sequences SEQ ID NO: 39, 31 and 40 respectively.

4. The method of claim 1, wherein each of said ABD comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3); and wherein:(i) the first ABD comprises:- a VH1 comprising a CDR-H1, H2 and H3 corresponding to the amino acid sequences of SEQ ID NO: 91 to 93 respectively, and- a VL1 comprising a CDR-L1, L2 and L3 corresponding to the amino acid sequences of SEQ ID NO: 97 to 99 respectively; and(ii) the second ABD comprises:- a VH2 comprising a CDR-H1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 13 to 15 respectively; the amino acid sequences of SEQ ID NO: 16 to 18 respectively; the amino acid sequences of SEQ ID NO: 19 to 21 respectively; the amino acid sequences of SEQ ID NO: 22 to 24 respectively; or the amino acid sequences of SEQ ID NO: 16, 25 and 26 respectively; and- a VL2 comprising a CDR-L1, 2 and 3 corresponding to: the amino acid sequences of SEQ ID NO: 27 to 29 respectively; the amino acid sequences of SEQ ID NO: 30 to 32 respectively; the amino acid sequences of SEQ ID NO: 33 to 35 respectively; the amino acid sequences of SEQ ID NO: 36 to 38 respectively; or the amino acid sequences SEQ ID NO: 39, 31 and 40 respectively.

5. The method of claim 1, wherein the binding protein comprises all or part of an immunoglobulin Fc region or variant thereof.

6. The method of claim 5, wherein all or part of the immunoglobulin Fc region or variant thereof binds to a human Fc-y receptor.

7. The method of claim 1, wherein the binding protein comprises three polypeptide chains(I), (II) and (III) that form two ABDs, as defined below:(I): light chain variable domain 1 A (VIA) - light chain constant domain 1 A (CIA) -Hingei- (CH2-CH3)A;(II): heavy chain variable domain IB (VIB) - heavy chain constant domain IB (CIB) - Hinge? - (CH2-CH3)B - LI - heavy chain variable domain 2A (V?A) - heavy chain constant domain 2 A (C2A) - Hinges; and(III): light chain variable domain 2B (V2B) - light chain constant domain 2B (C2B), wherein:VIA and VIB form a binding pair Vi (VHI / VLI);V2A and V2B form a binding pair V2 (VH2 / VL2);CIA and CIB form a pair Ci (CH1 / CL) and C2A and C2B form a pair C2 (CH1 / CL) wherein CHI is an immunoglobulin heavy chain constant domain 1 and CL is an immunoglobulin light chain constant domain;Hingei, Hinge2 and Hinges are identical or different and correspond to all or part of an immunoglobulin hinge region;(Cu2-CH3)Aand (CH2-CH3)B are identical or different, and comprise an immunoglobulin heavy chain constant domain 2 (CH2) and an immunoglobulin heavy chain constant domain 3 (CH3);LI is an amino acid linker.

8. The method of claim 7, wherein:CIB is an immunoglobulin heavy chain constant domain 1 (CHI);C2A is an immunoglobulin heavy chain constant domain 1 (CHI);CL corresponds to an immunoglobulin kappa light chain constant domain (CK); (CH2-CH3)A corresponds to the amino acid sequence of SEQ ID NO: 69;(CH2-CH3)B corresponds to the amino acid sequence of SEQ ID NO: 70;Hingei corresponds to the amino acid sequence of SEQ ID NO: 74;Hinge2 corresponds to the amino acid sequence of SEQ ID NO: 75;Hinges corresponds to the amino acid sequence of SEQ ID NO: 77;Li corresponds to the amino acid sequence of SEQ ID NO: 76.

9. The method of claim 5, wherein residue N297 of the Fc region or variant thereof according to EU numbering comprises a N-linked glycosylation.

10. The method of claim 5, wherein the all or part of the Fc region or variant thereof binds to a human CD16A (FcyRIII) polypeptide.11 The method of claim 5, wherein the Fc region or variant thereof is of an IgGl, IgG2, IgG3, or IgG4 isotype.

12. The method of claim 1, wherein the binding protein comprises at least two polypeptide chains linked by at least one disulfide bridge.

13. The method of claim 7, wherein the polypeptide chains (I) and (II) are linked by at least one disulfide bridge between CIA and Hinge? and / or wherein the polypeptide chains (II) and (III) are linked by at least one disulfide bridge between Hinges and C?B.

14. The method of claim 7, wherein VIA is VLI and VIB is VHL15. The method of claim 7, wherein V?A is VH2 and V2B is VL2.

16. The method of claim 2, wherein:(a) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 27; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 28; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29;(b) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32;(c) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 34; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35;(d) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 37; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38;(e) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 39; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40;(f) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 13; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 27; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 28; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29;(g) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32;(h) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 19; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35;(i) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 22; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38; or(j) VHI comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; VLI comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; VH2 comprises a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 39; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

17. The method of any claim 2, wherein:(a) VHI and VLI corresponds to the amino acid sequences of SEQ ID NO: 41 and 43 respectively or corresponds to the amino acid sequences of SEQ ID NO: 42 and 44 respectively; and / or(b) VH2 and VL2 corresponds 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;the amino acid sequences of SEQ ID NO: 51 and 59 respectively; or the amino acid sequences of SEQ ID NO: 52 and 60 respectively.

18. The method of claim 17, wherein:(a) VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 45; VL2 comprises the amino acid sequence of SEQ ID NO: 53;(b)Vm comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 46; VL2 comprises the amino acid sequence of SEQ ID NO: 54;(C)VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 47; VL2 comprises the amino acid sequence of SEQ ID NO: 55;(d)Vm comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 48; VL2 comprises the amino acid sequence of SEQ ID NO: 56;(e)Vm comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 49; VL2 comprises the amino acid sequence of SEQ ID NO: 57;(f) VHI comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 50; VL2 comprises the amino acid sequence of SEQ ID NO: 58;(g)Vm comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 51; VL2 comprises the amino acid sequence of SEQ ID NO: 59;(h)Vm comprises the amino acid sequence of SEQ ID NO: 41; VLI comprises the amino acid sequence of SEQ ID NO: 43; VH2 comprises the amino acid sequence of SEQ ID NO: 52; VL2 comprises the amino acid sequence of SEQ ID NO: 60;(i) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 45; VL2 comprises the amino acid sequence of SEQ ID NO: 53;(j) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 46; VL2 comprises the amino acid sequence of SEQ ID NO: 54;(k) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 47; VL2 comprises the amino acid sequence of SEQ ID NO: 55;(l) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 48; VL2 comprises the amino acid sequence of SEQ ID NO: 56;(m) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 49; VL2 comprises the amino acid sequence of SEQ ID NO: 57;(n) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 50; VL2 comprises the amino acid sequence of SEQ ID NO: 58;(o) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 51; VL2 comprises the amino acid sequence of SEQ ID NO: 59;(p) VHI comprises the amino acid sequence of SEQ ID NO: 42; VLI comprises the amino acid sequence of SEQ ID NO: 44; VH2 comprises the amino acid sequence of SEQ ID NO: 52; VL2 comprises the amino acid sequence of SEQ ID NO: 60.

19. The method of claim 7, wherein:- polypeptide (I) comprises an amino acid sequence of SEQ ID NO: 64;- polypeptide (II) comprises an amino acid sequence of SEQ ID NO: 65; and- polypeptide (III) comprises an amino acid sequence of SEQ ID NO: 66.

20. The method of claim 7, wherein:- polypeptide (I) consists of an amino acid sequence of SEQ ID NO: 64;- polypeptide (II) consists of an amino acid sequence of SEQ ID NO: 65; and- polypeptide (III) consists of an amino acid sequence of SEQ ID NO: 66.

21. The method of claim 1, wherein the IL-2 polypeptide comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 84.

22. The method of claim 1, wherein the IL-2 polypeptide comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 85.

23. The method of claim 1, wherein the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 85 in which at least one amino acid residue is replaced by an unnatural amino acid.

24. The method of claim 23, wherein the position of the at least one unnatural amino acid is selected from K34, T36, R37, T40, F41, K42, F43, Y44, E59, E60, E61, K63, P64, E67, V68, N70, L71, M103, C104, and Y107, wherein the residue positions correspond to the positions 34, 36, 37, 40, 41, 42, 43, 44, 60, 61, 63, 64, 67, 68, 70, 71, 103, 104, and 106 as set forth in SEQ ID NO: 85.

25. The method of claim 23, wherein the position of the at least one unnatural amino acid is selected from T36, R37, T40, F41, K42, F43, Y44, E60, E61, P64, E67, and L71 , wherein the residue positions correspond to the positions 36, 37, 40, 41, 42, 43, 44, 60, 61, 64, 67, and 71 as set forth in SEQ ID NO: 85.

26. The method of claim 23, wherein the position of the at least one unnatural amino acid is selected from K34, K63, V68, N70, M103, C104, and Y106, wherein the residue positions correspond to the positions 34, 63, 68, 70, 103, 104, and 106 as set forth in SEQ ID NO: 85.

27. The method of claim 23, wherein the position of the at least one unnatural amino acid is selected from T36, R37, T40, Y44, E60, E67, and L71, wherein the residue positions correspond to the positions 36, 37, 40, 44, 60, 67, and 71 as set forth in SEQ ID NO: 85.

28. The method of claim 23, wherein the position of the at least one unnatural amino acid is selected from F41, K42, F43, E61, and P64, wherein the residue positions correspond to the positions 41, 42, 43, 61, and 64 as set forth in SEQ ID NO: 85.

29. The method of claim 23, wherein the at least one unnatural amino acid:• is a lysine analogue;• comprises an aromatic side chain;• comprises an azido group;• comprises an alkyne group; or• comprises an aldehyde or ketone group.

30. The method of claim 23, wherein the at least one unnatural amino acid does not comprise an aromatic side chain.

31. The method of claim 23, wherein the at least one unnatural amino acid comprises N6- azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbomene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine, 2-amino-8- oxononanoic acid, 2-amino-8-oxooctanoic acid, p- acetyl-L-phenylalanine, p-azidomethyl-L- phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8- oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl- phenylalanine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L- phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p- amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, 0- 4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L- phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3- oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic, or selenocysteine.

32. The method of claim 23, wherein the IL-2 polypeptide is covalently attached to a conjugating moiety through the at least one unnatural amino acid.

33. The method of claim 32, wherein the conjugating moiety comprises a water-soluble polymer, a lipid, a protein, or a peptide.

34. The method of claim 32, wherein the water-soluble polymer comprises polyethylene glycol (PEG), polypropylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N- acryloylmorpholine), or a combination thereof.

35. The method of claim 32, wherein the water-soluble polymer comprises a PEG molecule.

36. The method of claim 34, wherein the PEG molecule is a linear PEG.

37. The method of claim 34, wherein the PEG molecule is a branched PEG.

38. The method of claim 32, wherein the water-soluble polymer comprises a polysaccharide.

39. The method of claim 38, wherein the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxy ethylstarch (EES).

40. The method of claim 33, wherein the lipid comprises a fatty acid.

41. The method of claim 40, wherein the fatty acid comprises from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms.

42. The method of claim 40, wherein the fatty acid is a saturated fatty acid.

43. The method of claim 33, wherein the protein comprises an albumin, a transferrin, or a transthyretin.

44. The method of claim 32, wherein the conjugating moiety comprises a toll-like receptor (TLR) agonist.

45. The method of claim 32, wherein the conjugating moiety is indirectly bound to the at least one unnatural amino acid of the IL-2 polypeptide through a linker.

46. The method of claim 45, wherein the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or a non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof.

47. The method of claim 19, wherein the IL-2 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof.

48. The method of claim 47, wherein the N-terminal deletion comprises a deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from the N-terminus, wherein the residue positions are in reference to the positions in SEQ ID NO: 85.

49. The method of claim 47, wherein the C-terminal deletion comprises a deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues from the C-terminus, wherein the residue positions are in reference to the positions in SEQ ID NO: 85.

50. The method of claim 32, wherein the conjugating moiety or the unnatural amino acid impairs or blocks the binding of IL-2 with IL-2Ra.

51. The method of claim 1, wherein the IL-2 polypeptide comprises an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 85, wherein the amino acid at positionP64 is replaced by the structure of Formula (I):Formula (I) wherein:W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; q is 1, 2, or 3;X has the structure:wherein X-l indicates the point of attachment to the preceding amino acid residue; and wherein X+l indicates the point of attachment to the following amino acid residue.

52. The method of claim 51, wherein the PEG group has an average molecular weight of about 30 kDa.

53. The method of claim 51, wherein Z is CH2 and Y is54. The method of claim 51, wherein Y is CH2 and Z is55. The method of claim 51, wherein56. The method of claim 51, wherein57. The method of claim 51, wherein the IL-2 conjugate comprises (i) an IL-2 conjugate in which the structure of Formula (I) has the structure of Formula (IV), and / or (ii) an IL-2 conjugate in which the structure of Formula (I) has the structure of or Formula (V):Formula (V); wherein: q is 1, 2, or 3;X has the structure:wherein X-l indicates the point of attachment to the preceding amino acid residue; and wherein X+l indicates the point of attachment to the following amino acid residue.

58. The method of claim 51, wherein the IL-2 conjugate comprises (i) an IL-2 conjugate in which the structure of Formula (I) has the structure of Formula (XII), and / or (ii) an IL-2 conjugate in which the structure of Formula (I) has the structure of or Formula (XIII):Formula (XIII); wherein: n is an integer such that a PEG group having the structure of -(OCEECEEjn-OCHa has a molecular weight of about 30 kDa; q is 1, 2, or 3; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 85 that are not replaced.

59. The method of claim 51, wherein q is 1.

60. The method of claim 51, wherein q is 2.

61. The method of claim 51, wherein q is 3.

62. The method of claim 31, wherein the IL-2 polypeptide that is covalently attached to a conjugating moiety has reduced binding between the IL-2 polypeptide and interleukin 2 receptor alpha (IL-2Ra) but retains significant binding with interleukin 2 Py receptor (IL-2R Py) signaling complex to form an IL-2 / IL- 2RPy complex, wherein the reduced binding to IL- 2Ra is compared to binding between a wild-type IL-2 polypeptide and IL-2Ra.

63. The method of claim 62, wherein the reduced binding is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% reduced binding to IL-2Ra relative to a wild-type IL-2 polypeptide.

64. The method of claim 62, wherein the reduced binding is about 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold, or more reduced binding to IL-2Ra relative to a wild-type IL-2 polypeptide.

65. The method of claim 1, wherein the binding protein is administered to the subject prior to the IL-2 polypeptide.

66. The method of claim 1, wherein the IL-2 polypeptide is administered to the subject prior to the binding protein.

67. The method of claim 1, wherein the binding protein and the IL-2 polypeptide thereof are administered contemporaneously to the subject.

68. The method of claim 1, wherein the cancer is a blood cancer.

69. The method of claim 1, wherein the cancer is myelodysplastic syndrome (MDS) or a lymphoproliferative disorder.

70. The method of claim 1, wherein the cancer is Acute Myeloid Leukemia (AML).

71. The method of claim 1, wherein cytotoxicity of cancer cells in the subject is induced after administration of the binding protein and the IL-2 polypeptide.

72. The method of claim 1, wherein NK cell activation in the subject is induced after administration of the binding protein and the IL-2 polypeptide.

73. The method of claim 72, wherein NK cell activation is determined by measuringCD69 expression from NK cells in the subject.

74. The method of any one of claim 1, wherein administration of the binding protein and the IL-2 polypeptide to the subject results in increased release of one or more effector cytokines.

75. The method of claim 74, wherein the one or more effector cytokines are IFN-y, TNF- a, MIP-la, MIP-ip, Granzyme A, Granzyme B, and Lymphotactin.

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