Uses of nk cell engaging antibody fusion constructs for treatments

Abstract

The invention relates to multispecific antigen-binding proteins for engaging natural killer (NK) cells for triggering NK cell cytotoxicity by engaging the CD16A (FcγRIIIA)expressed on NK cells, wherein the antigen-binding protein comprises at least two CD16A antigen-binding moieties and at least a further target antigen-binding moiety. The CD16A antigen-binding moiety comprises light chain and heavy chain variable regions linked one after another in a polypeptide chain and the variable region at the N-terminus of the polypeptide chain comprising the CD16A antigen-binding moiety is a light chain variable region.

What is claimed is: 
     
         1 . A method of treating a B cell maturation antigen (BCMA)-expressing cancer in a patient comprising administering a therapeutically effective amount of a multispecific antigen-binding protein, wherein said multispecific antigen-binding protein comprises:
 a) two BCMA antigen-binding moieties each comprising a Fab fragment and an Fc portion and wherein each Fab fragment comprises: a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:67, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:68, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:69; and a light chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:70, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:71, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:72, and   b) two CD16A antigen-binding moieties each in the format of a single chain variable fragment (scFv) comprising: a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:73, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:74, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:75; and a light chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:76, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:77, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:78, wherein the variable region at the N-terminus of each of the two scFvs is the light chain variable region,   
       wherein the N-terminus of the first of the two scFvs is fused to the C-terminus of the Fc portion of the first of the two BCMA binding moieties and the N-terminus of the second of the two scFvs is fused to the C-terminus of the Fc portion of the second of the two BCMA binding moieties. 
     
     
         2 . The method of  claim 1 , wherein the multispecific antigen-binding protein comprises an Fc portion mutation selected from the group consisting of (a) C220S, C229S, E233P, L234A, L234V, L234F, L235A, L235E, P238S, D265A, N297A, N297Q, and P331S, or (b) L234A, L234V, L234F, L235A, L235E, P238S, and D265A, according to the Kabat EU numbering. 
     
     
         3 . The method of  claim 1 , wherein the two CD16A antigen-binding moieties of the multispecific antigen-binding protein each comprise:
 a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3; and/or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2.   
     
     
         4 . The method of  claim 1 , wherein the multispecific antigen-binding protein is a tetramer comprising a first polypeptide chain consisting of the amino acid sequence set forth in SEQ ID NO:
 61, and a second polypeptide chain consisting of the amino acid sequence set forth in SEQ ID NO: 62.   
     
     
         5 . The method of  claim 1 , wherein the two BCMA antigen-binding moieties each comprise: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65 and/or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66. 
     
     
         6 . The method of  claim 1 , wherein the BCMA-expressing cancer is multiple myeloma. 
     
     
         7 . A method of treating a BCMA-expressing cancer in a patient comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a multispecific antigen-binding protein comprising two CD16A antigen-binding moieties, wherein each CD16A antigen-binding moiety is in the format of a single chain variable fragment (scFv), and two B cell maturation antigen (BCMA) antigen-binding moieties, wherein each BCMA antigen-binding moiety comprises an Fab fragment and an Fc portion, wherein:
 (i) the two CD16A antigen-binding moieties each comprise:
 (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:73, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:74, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:75; and 
 (b) a light chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:76, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:77, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:78; wherein the variable region at the N-terminus of each of the two scFvs is the light chain variable region; 
 (c) wherein the N-terminus of the first of the two scFvs is fused to the C-terminus of the Fc portion of the first of the two BCMA binding moieties and the N-terminus of the second of the two scFvs is fused to the C-terminus of the Fc portion of the second of the two BCMA binding moieties; and 
   (ii) the two BCMA antigen-binding moieties each comprise:
 (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:67, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:68, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:69; and 
 (b) a light chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:70, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:71, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:72; 
   
       and a pharmaceutically acceptable carrier. 
     
     
         8 . The method of  claim 7 , wherein the BCMA-expressing cancer is multiple myeloma. 
     
     
         9 . A method of treating a BCMA-expressing cancer in a patient comprising administering a therapeutically effective amount of a bispecific antigen-binding protein comprising two CD16A antigen-binding moieties each in the format of a single chain variable fragment (scFv) and two B cell maturation antigen (BCMA)-targeting moieties each comprising an Fab fragment and an Fc portion, wherein the N-terminus of the first of the two scFvs is fused to the C-terminus of the first of the two BCMA-targeting moieties, and the N-terminus of the second of the two scFvs is fused to the C-terminus of the second of the two BCMA-targeting moieties wherein:
 (i) the two CD16A antigen-binding moieties each comprise:
 (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:73; a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:74; and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:75, and 
 (b) a light chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:76; a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:77; and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:78; wherein the variable region at the N-terminus of each of the two scFvs is the light chain variable region; and 
   (ii) the two BCMA antigen-binding moieties each comprise:
 (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:67; a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:68; and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:69, and 
   (ii) a light chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:70; a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:71; and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:72.   
     
     
         10 . A method of  claim 9 , wherein:
 (i) the two CD16A antigen-binding moieties each comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3;   (ii) the two CD16A antigen-binding moieties each comprise a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2;   (iii) the two BCMA antigen-binding moieties each comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65;   (iv) the two BCMA antigen-binding moieties each comprise a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66;   (v) the two CD16A antigen-binding moieties each comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2;   (vi) the two BCMA antigen-binding moieties each comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66; and/or   (vii) the two CD16A antigen-binding moieties each comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; and the two BCMA antigen-binding moieties each comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66.   
     
     
         11 . The method of  claim 9 , wherein the bispecific antigen-binding protein comprises an Fc portion mutation selected from the group consisting of (a) C220S, C229S, E233P, L234A, L234V, L234F, L235A, L235E, P238S, D265A, N297A, N297Q, and P331S, or (b) L234A, L234V, L234F, L235A, L235E, P238S, and D265A, according to the Kabat EU numbering. 
     
     
         12 . The method of  claim 9 , wherein the BCMA-expressing cancer is multiple myeloma. 
     
     
         13 . A method of treating a BCMA-expressing cancer in a subject having a depleted or reduced NK cell population comprising administering a therapeutically effective amount of a multispecific antigen-binding protein, wherein said multispecific antigen-binding protein comprises:
 c) two B cell maturation antigen (BCMA) antigen-binding moieties each comprising a Fab fragment and an Fc portion and wherein each Fab fragment comprises: a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:67, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:68, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:69; and a light chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:70, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:71, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:72, and   d) two CD16A antigen-binding moieties each in the format of a single chain variable fragment (scFv) comprising: a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:73, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:74, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:75; and a light chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:76, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:77, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:78, wherein the variable region at the N-terminus of each of the two scFvs is the light chain variable region,   
       wherein the N-terminus of the first of the two scFvs is fused to the C-terminus of the Fc portion of the first of the two BCMA binding moieties and the N-terminus of the second of the two scFvs is fused to the C-terminus of the Fc portion of the second of the two BCMA binding moieties. 
     
     
         14 . The method of  claim 13 , wherein the multispecific antigen-binding protein comprises an Fc portion mutation selected from the group consisting of (a) C220S, C229S, E233P, L234A, L234V, L234F, L235A, L235E, P238S, D265A, N297A, N297Q, and P331S, or (b) L234A, L234V, L234F, L235A, L235E, P238S, and D265A, according to the Kabat EU numbering. 
     
     
         15 . The method of  claim 13 , wherein the two CD16A antigen-binding moieties of the multispecific antigen-binding protein each comprise:
 a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:   3; and/or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2.   
     
     
         16 . The method of  claim 13 , wherein the multispecific antigen-binding protein is a tetramer comprising a first polypeptide chain consisting of the amino acid sequence set forth in SEQ ID NO: 61, and a second polypeptide chain consisting of the amino acid sequence set forth in SEQ ID NO: 62. 
     
     
         17 . The method of  claim 13 , wherein the two BCMA antigen-binding moieties each comprise: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65 and/or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66. 
     
     
         18 . The method of  claim 13 , wherein the BCMA-expressing cancer is multiple myeloma. 
     
     
         19 . A method of treating a BCMA-expressing cancer in a subject having a depleted or reduced NK cell population comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a multispecific antigen-binding protein comprising two CD16A antigen-binding moieties, wherein each CD16A antigen-binding moiety is in the format of a single chain variable fragment (scFv), and two B cell maturation antigen (BCMA) antigen-binding moieties, wherein each BCMA antigen-binding moiety comprises an Fab fragment and an Fc portion, wherein:
 (i) the two CD16A antigen-binding moieties each comprise:
 (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:73, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:74, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:75; and 
 (b) a light chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:76, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:77, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:78; wherein the variable region at the N-terminus of each of the two scFvs is the light chain variable region; 
 (c) wherein the N-terminus of the first of the two scFvs is fused to the C-terminus of the Fc portion of the first of the two BCMA binding moieties and the N-terminus of the second of the two scFvs is fused to the C-terminus of the Fc portion of the second of the two BCMA binding moieties; and 
   (ii) the two BCMA antigen-binding moieties each comprise:
 (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:67, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:68, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:69; and 
 (b) a light chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:70, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:71, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:72; 
   
       and a pharmaceutically acceptable carrier. 
     
     
         20 . The method of  claim 19 , wherein the BCMA-expressing cancer is multiple myeloma. 
     
     
         21 . A method of treating a BCMA-expressing cancer in a subject having a depleted or reduced NK cell population comprising administering a therapeutically effective amount of a bispecific antigen-binding protein comprising two CD16A antigen-binding moieties each in the format of a single chain variable fragment (scFv) and two B cell maturation antigen (BCMA)-targeting moieties each comprising an Fab fragment and an Fc portion, wherein the N-terminus of the first of the two scFvs is fused to the C-terminus of the first of the two BCMA-targeting moieties, and the N-terminus of the second of the two scFvs is fused to the C-terminus of the second of the two BCMA-targeting moieties wherein:
 (i) the two CD16A antigen-binding moieties each comprise:
 (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:73; a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:74; and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:75, and 
 (b) a light chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:76; a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:77; and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:78; wherein the variable region at the N-terminus of each of the two scFvs is the light chain variable region; and 
   (ii) the two BCMA antigen-binding moieties each comprise:
 (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:67; a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:68; and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:69, and 
 (ii) a light chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:70; a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:71; and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:72. 
   
     
     
         22 . A method of  claim 21 , wherein:
 (i) the two CD16A antigen-binding moieties each comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3;   (ii) the two CD16A antigen-binding moieties each comprise a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2;   (iii) the two BCMA antigen-binding moieties each comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65;   (iv) the two BCMA antigen-binding moieties each comprise a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66;   (v) the two CD16A antigen-binding moieties each comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2;   (vi) the two BCMA antigen-binding moieties each comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66; and/or   (vii) the two CD16A antigen-binding moieties each comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; and the two BCMA antigen-binding moieties each comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66.   
     
     
         23 . The method of  claim 22 , wherein the bispecific antigen-binding protein comprises an Fc portion mutation selected from the group consisting of (a) C220S, C229S, E233P, L234A, L234V, L234F, L235A, L235E, P238S, D265A, N297A, N297Q, and P331S, or (b) L234A, L234V, L234F, L235A, L235E, P238S, and D265A, according to the Kabat EU numbering. 
     
     
         24 . The method of  claim 22 , wherein the BCMA-expressing cancer is multiple myeloma.