MULTIFUNCTIONAL NATURAL KILLER (NK) CELL ENGAGERS BINDING TO NKp46 AND CD123

Abstract

The present disclosure relates to multifunctional binding proteins comprising a first and a second antigen binding domains (ABDs) and all or part of an immunoglobulin Fc region or variant thereof, wherein the first ABD binds specifically to human CD123 and the second ABD binds specifically to human NKp46 and wherein all or part of the immunoglobulin Fc region or variant thereof to a human Fc-γ receptor. The disclosure also relates to methods for making said binding proteins, compositions thereof, and their uses, including the treatment or prevention of proliferative disorders, including Acute Myeloid Leukemia (AML) and myelodysplastic syndromes (MDS).

1 - 19 . (canceled) 
     
     
         20 . An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a binding protein comprising a first and a second antigen binding domain (ABD) and all or part of an immunoglobulin Fc region or variant thereof, 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 binds specifically to human CD123 and 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; 
   (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
 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;   and wherein all or part of the immunoglobulin Fc region or variant thereof binds to a human Fc-γ receptor.   
     
     
         21 . An expression vector comprising the nucleic acid molecule of  claim 20 . 
     
     
         22 . An isolated cell comprising the nucleic acid molecule of  claim 20 . 
     
     
         23 . An isolated cell comprising the expression vector of  claim 21 . 
     
     
         24 . The isolated cell of  claim 23 , wherein the host cell is a mammalian cell. 
     
     
         25 . A method for making the binding protein according to  claim 20 , comprising a step of:
 (a) culturing host cell(s) under conditions suitable for expressing a plurality of recombinant polypeptides, said plurality comprising (i) a polypeptide comprising an amino acid sequence of SEQ ID NO: 64, and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO: 65, and (iii) a polypeptide comprising an amino acid sequence of SEQ ID NO: 66;   (b) optionally recovering the expressed recombinant polypeptides.   
     
     
         26 . A method of treating or preventing a blood cancer, the method comprising administering to a subject in need of said treatment or prevention a binding protein comprising a first and a second antigen binding domain (ABD) and all or part of an immunoglobulin Fc region or variant thereof, wherein the first ABD binds specifically to human CD123, the second ABD binds specifically to human NKp46, and wherein all or part of the immunoglobulin Fc region or variant thereof binds to a human Fc-γ receptor. 
     
     
         27 . A method of treating or preventing a myelodysplastic syndrome (MDS) or a lymphoproliferative disorder, the method comprising administering to a subject in need of said treatment or prevention a binding protein comprising a first and a second antigen binding domain (ABD) and all or part of an immunoglobulin Fc region or variant thereof, wherein the first ABD binds specifically to human CD123, the second ABD binds specifically to human NKp46, and wherein all or part of the immunoglobulin Fc region or variant thereof binds to a human Fc-γ receptor. 
     
     
         28 . A method of treating or preventing an Acute Myeloid Leukemia (AML), the method comprising administering to a subject in need of said treatment or prevention a binding protein comprising a first and a second antigen binding domain (ABD) and all or part of an immunoglobulin Fc region or variant thereof, wherein the first ABD binds specifically to human CD123, the second ABD binds specifically to human NKp46, and wherein all or part of the immunoglobulin Fc region or variant thereof binds to a human Fc-γ receptor. 
     
     
         29 . (canceled) 
     
     
         30 . A method of treating or preventing a CD64-positive Acute Myeloid Leukemia (AML), the method comprising administering to a subject in need of said treatment or prevention a binding protein comprising a first and a second antigen binding domain (ABD) and all or part of an immunoglobulin Fc region or variant thereof, wherein the first ABD binds specifically to human CD123, the second ABD binds specifically to human NKp46, and wherein all or part of the immunoglobulin Fc region or variant thereof binds to a human Fc-γ receptor. 
     
     
         31 . The nucleic acid molecule according to  claim 20 , wherein the binding protein comprises three polypeptide chains (I), (II) and (III) that form two ABDs, as defined below:
   V 1A -C 1A -Hinge 1 -(C H 2-C H 3) A   (I)
     V 1B -C 1B -Hinge 2 -(C H 2-C H 3) B -L 1 -V 2A -C 2A -Hinge 3   (II)
     V 2B -C 2B   (III)
   
       wherein:
 V 1A  and V 1B  form a binding pair V 1  (V H1 /V L1 ); 
 V 2A  and V 2B  form a binding pair V 2  (V H2 /V L2 ); 
 C 1A  and C 1B  form a pair C 1  (C H 1/C L ) and C 2A  and C 2B  form a pair C 2  (C H 1/C L ) wherein C H 1 is an immunoglobulin heavy chain constant domain 1 and C L  is an immunoglobulin light chain constant domain; 
 Hinge 1 , Hinge 2  and Hinge 3  are identical or different and correspond to all or part of an immunoglobulin hinge region; 
 (C H 2-C H 3) A  and (C H 2-C H 3) B  are identical or different, and comprise an immunoglobulin heavy chain constant domain 2 (C H 2) and an immunoglobulin heavy chain constant domain 3 (C H 3); 
 L 1  is an amino acid linker. 
 
     
     
         32 . The nucleic acid according to  claim 31 , wherein:
 C 1B  is an immunoglobulin heavy chain constant domain 1 (C H 1);   C 2A  is an immunoglobulin heavy chain constant domain 1 (C H 1);   C L  corresponds to an immunoglobulin kappa light chain constant domain (C κ );   (C H 2-C H 3) A  corresponds to the amino acid sequence of SEQ ID NO: 69;   (C H 2-C H 3) B  corresponds to the amino acid sequence of SEQ ID NO: 70;   Hinge 1  corresponds to the amino acid sequence of SEQ ID NO:74;   Hinge 2  corresponds to the amino acid sequence of SEQ ID NO:75;   Hinge 3  corresponds to the amino acid sequence of SEQ ID NO: 77;   L 1  corresponds to the amino acid sequence of SEQ ID NO: 76.   
     
     
         33 . The nucleic acid according to  claim 20 , wherein the residue N297 of the Fc region or variant thereof according to EU numbering comprises a N-linked glycosylation. 
     
     
         34 . The nucleic acid according to  claim 20 , wherein all or part of the Fc region or variant thereof binds to a human CD16A ((FcγRIII) polypeptide. 
     
     
         35 . The nucleic acid according to  claim 20 , wherein the binding protein comprises at least two polypeptide chains linked by at least one disulfide bridge. 
     
     
         36 . The nucleic acid according to  claim 35 , wherein the polypeptide chains (I) and (II) are linked by at least one disulfide bridge between C 1A  and Hinge 2  and/or wherein the polypeptide chains (II) and (III) are linked by at least one disulfide bridge between Hinge 3  and C 2B . 
     
     
         37 . The nucleic acid according to  claim 31 , wherein V 1A  is V L1  and V 1B  is V H1 . 
     
     
         38 . The nucleic acid according to  claim 31 , wherein V 2A  is V H2  and V 2B  is V L2 . 
     
     
         39 . The nucleic acid according to  claim 20 , wherein:
 (a) V H1  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; V L1  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; V H2  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; V L2  comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 27; a CDR-L 2  comprising the amino acid sequence of SEQ ID NO: 28; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29;   (b) V H1  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; V L1  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; V H2  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; V L2  comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30; a CDR-L 2  comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32;   (c) V H1  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; V L1  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; V H2  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; V L2  comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33; a CDR-L 2  comprising the amino acid sequence of SEQ ID NO: 34; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35;   (d) V H1  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; V L1  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; V H2  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; V L2  comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36; a CDR-L 2  comprising the amino acid sequence of SEQ ID NO: 37; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38;   (e) V H1  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; V L1  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; V H2  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; V L2  comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 39; a CDR-L 2  comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40;   (f) V H1  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; V L1  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; V H2  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; V L2  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) V H1  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; V L1  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; V H2  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; V L2  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) V H1  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; V L1  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; V H2  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; V L2  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) V H1  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; V L1  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; V H2  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; V L2  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) V H1  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; V L1  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; V H2  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; V L2  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.   
     
     
         40 . The nucleic acid according to  claim 20 , wherein:
 (a) V H1  and V L1  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) V H2  and V L2  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.