Dll3-targeting multispecific antigen-binding molecules and uses thereof

Abstract

The disclosure provides multispecific antigen-binding molecules that comprise a first antigen-binding moiety and a second antigen-binding moiety, each of which is capable of binding to CD3 and CD137, but does not bind to CD3 and CD137 at the same time; and a third antigen-binding moiety that is capable of binding to DLL3, preferably human DLL3, which induce T-cell dependent cytotoxity more efficiently whilst circumventing adverse toxicity concerns or side effects that other multispecific antigen-binding molecules may have. The present invention provides multispecific antigen-binding molecules and pharmaceutical compositions that can treat various cancers, especially those associated with DLL3, by comprising the antigen-binding molecule as an active ingredient.

1 . A multispecific antigen-binding molecule comprising:
 a first antigen-binding moiety and a second antigen-binding moiety, each of which is capable of binding to CD3 and CD137, but does not bind to CD3 and CD137 at the same time; and   a third antigen-binding moiety that is capable of binding to DLL3, preferably human DLL3.   
     
     
         2 . The multispecific antigen-binding molecule of  claim 1 , wherein the first antigen-binding moiety and the second antigen-binding moiety each comprises an antibody variable region comprising any one of (a1) to (a17) below:
 (a1) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 17, the heavy chain CDR 2 of SEQ ID NO: 31, the heavy chain CDR 3 of SEQ ID NO: 45, the light chain CDR 1 of SEQ ID NO: 64, the light chain CDR 2 of SEQ ID NO: 69 and the light chain CDR 3 of SEQ ID NO: 74;   (a2) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 18, the heavy chain CDR 2 of SEQ ID NO: 32, the heavy chain CDR 3 of SEQ ID NO: 46, the light chain CDR 1 of SEQ ID NO: 63, the light chain CDR 2 of SEQ ID NO: 68 and the light chain CDR 3 of SEQ ID NO: 73;   (a3) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 19, the heavy chain CDR 2 of SEQ ID NO: 33, the heavy chain CDR 3 of SEQ ID NO: 47, the light chain CDR 1 of SEQ ID NO: 63, the light chain CDR 2 of SEQ ID NO: 68 and the light chain CDR 3 of SEQ ID NO: 73;   (a4) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 19, the heavy chain CDR 2 of SEQ ID NO: 33, the heavy chain CDR 3 of SEQ ID NO: 47, the light chain CDR 1 of SEQ ID NO: 65, the light chain CDR 2 of SEQ ID NO: 70 and the light chain CDR 3 of SEQ ID NO: 75;   (a5) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 20, the heavy chain CDR 2 of SEQ ID NO: 34, the heavy chain CDR 3 of SEQ ID NO: 48, the light chain CDR 1 of SEQ ID NO: 63, the light chain CDR 2 of SEQ ID NO: 68 and the light chain CDR 3 of SEQ ID NO: 73;   (a6) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 22, the heavy chain CDR 2 of SEQ ID NO: 36, the heavy chain CDR 3 of SEQ ID NO: 50, the light chain CDR 1 of SEQ ID NO: 63, the light chain CDR 2 of SEQ ID NO: 68 and the light chain CDR 3 of SEQ ID NO: 73;   (a7) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 23, the heavy chain CDR 2 of SEQ ID NO: 37, the heavy chain CDR 3 of SEQ ID NO: 51, the light chain CDR 1 of SEQ ID NO: 63, the light chain CDR 2 of SEQ ID NO: 68 and the light chain CDR 3 of SEQ ID NO: 73;   (a8) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 23, the heavy chain CDR 2 of SEQ ID NO: 37, the heavy chain CDR 3 of SEQ ID NO: 51, the light chain CDR 1 of SEQ ID NO: 66, the light chain CDR 2 of SEQ ID NO: 71 and the light chain CDR 3 of SEQ ID NO: 76;   (a9) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 24, the heavy chain CDR 2 of SEQ ID NO: 38, the heavy chain CDR 3 of SEQ ID NO: 52, the light chain CDR 1 of SEQ ID NO: 63, the light chain CDR 2 of SEQ ID NO: 68 and the light chain CDR 3 of SEQ ID NO: 73;   (a10) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 25, the heavy chain CDR 2 of SEQ ID NO: 39, the heavy chain CDR 3 of SEQ ID NO: 53, the light chain CDR 1 of SEQ ID NO: 66, the light chain CDR 2 of SEQ ID NO: 71 and the light chain CDR 3 of SEQ ID NO: 76;   (a11) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 26, the heavy chain CDR 2 of SEQ ID NO: 40, the heavy chain CDR 3 of SEQ ID NO: 54, the light chain CDR 1 of SEQ ID NO: 66, the light chain CDR 2 of SEQ ID NO: 71 and the light chain CDR 3 of SEQ ID NO: 76;   (a12) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 26, the heavy chain CDR 2 of SEQ ID NO: 40, the heavy chain CDR 3 of SEQ ID NO: 54, the light chain CDR 1 of SEQ ID NO: 63, the light chain CDR 2 of SEQ ID NO: 68 and the light chain CDR 3 of SEQ ID NO: 73;   (a13) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 27, the heavy chain CDR 2 of SEQ ID NO: 41, the heavy chain CDR 3 of SEQ ID NO: 55, the light chain CDR 1 of SEQ ID NO: 63, the light chain CDR 2 of SEQ ID NO: 68 and the light chain CDR 3 of SEQ ID NO: 73;   (a14) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 28, the heavy chain CDR 2 of SEQ ID NO: 42, the heavy chain CDR 3 of SEQ ID NO: 56, the light chain CDR 1 of SEQ ID NO: 63, the light chain CDR 2 of SEQ ID NO: 68 and the light chain CDR 3 of SEQ ID NO: 73;   (a15) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 82, the heavy chain CDR 2 of SEQ ID NO: 83, the heavy chain CDR 3 of SEQ ID NO: 84, the light chain CDR 1 of SEQ ID NO: 65, the light chain CDR 2 of SEQ ID NO: 70 and the light chain CDR 3 of SEQ ID NO: 75;   (a16) an antibody variable region that binds to the same epitope of any of the antibody variable region selected from (a1) to (a15); and   (a17) an antibody variable fragment that competes with the binding of any of the antibody variable fragment selected from (a1) to (a15).   
     
     
         3 . The multispecific antigen-binding molecule of any one of  claims 1  to  2 , wherein the first antigen-binding moiety and the second antigen-binding moiety each comprises an antibody variable region comprising any one of (a1) to (a17) below:
 (a1) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 59; 
 (a2) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 4, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58; 
 (a3) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 5, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58; 
 (a4) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 5, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 60; 
 (a5) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 6, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58; 
 (a6) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 8, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58; 
 (a7) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58; 
 (a8) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61; 
 (a9) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58; 
 (a10) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61; 
 (a11) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61; 
 (a12) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58; 
 (a13) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58; 
 (a14) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 14, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58; 
 (a15) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 81, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 60; 
 (a16) an antibody variable region that binds to the same epitope of any of the antibody variable region selected from (a1) to (a15); and 
 (a17) an antibody variable fragment that competes with the binding of any of the antibody variable fragment selected from (a1) to (a15). 
 
     
     
         4 . The multispecific antigen-binding molecule of any one of  claims 1  to  3 , wherein each of the first antigen-binding moiety and the second antigen-binding moiety is a Fab molecule and comprises at least one disulfide bond formed between the CH1 region of the first antigen-binding moiety and the CH1 region of the second antigen-binding moiety. 
     
     
         5 . The multispecific antigen-binding molecule of  claim 4 , wherein each of the first antigen-binding moiety and the second antigen-binding moiety is a Fab molecule and comprises one disulfide bond formed between the amino acid residues at position 191 according to EU numbering in the respective CH1 region of the first antigen-binding moiety and the second antigen-binding moiety. 
     
     
         6 . The multispecific antigen-binding molecule of any one of  claims 1  to  5 , wherein each of the first, second and third antigen binding moiety is a Fab molecule, wherein the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain (CH1) to the N-terminus of the Fab heavy chain of either one of the first antigen binding moiety or the second antigen binding moiety, optionally via a peptide linker. 
     
     
         7 . The multispecific antigen-binding molecule of  claim 6 , wherein said peptide linker is selected from the group consisting of the amino acid sequence of SEQ ID NO: 248, SEQ ID NO: 249 or SEQ ID NO: 259. 
     
     
         8 . The multispecific antigen-binding molecule of any one of  claims 1  to  7 , wherein the third antigen binding moiety is a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and wherein each of the first and second antigen binding moiety is a conventional Fab molecule. 
     
     
         9 . The multispecific antigen-binding molecule of  claim 8 , wherein in the constant domain CL of the light chain of each of the first and second antigen binding moiety, the amino acids at position 123 and 124 are arginine (R) and lysine (K) respectively (numbering according to Kabat), and wherein in the constant domain CH1 of the heavy chain of each of the first and second antigen binding moiety the amino acids at position 147 and 213 are glutamic acid (E) (numbering according to Kabat EU index). 
     
     
         10 . The multispecific antigen-binding molecule of any one of  claims 1  to  9 , wherein the third antigen-binding moiety capable of binding to DLL3 comprises an antibody variable region comprising any one of (a1) to (a5) below:
 (a1) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 233, the heavy chain CDR 2 of SEQ ID NO: 234, the heavy chain CDR 3 of SEQ ID NO: 235, the light chain CDR 1 of SEQ ID NO: 237, the light chain CDR 2 of SEQ ID NO: 238 and the light chain CDR 3 of SEQ ID NO: 239; 
 (a2) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 276, the heavy chain CDR 2 of SEQ ID NO: 277, the heavy chain CDR 3 of SEQ ID NO: 278, the light chain CDR 1 of SEQ ID NO: 279, the light chain CDR 2 of SEQ ID NO: 280 and the light chain CDR 3 of SEQ ID NO: 281; 
 (a3) the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 285, the heavy chain CDR 2 of SEQ ID NO: 286, the heavy chain CDR 3 of SEQ ID NO: 287, the light chain CDR 1 of SEQ ID NO: 288, the light chain CDR 2 of SEQ ID NO: 289 and the light chain CDR 3 of SEQ ID NO: 290; 
 (a4) an antibody variable region that binds to the same epitope of any of the antibody variable region selected from (a1) to (a3); and 
 (a5) an antibody variable fragment that competes with the binding of any of the antibody variable fragment selected from (a1) to (a3). 
 
     
     
         11 . The multispecific antigen-binding molecule of any one of  claims 1  to  10 , wherein the third antigen-binding moiety capable of binding to DLL3 comprises an antibody variable region comprising any one of (a1) to (a6) below:
 (a1) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 232, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 236; 
 (a2) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 264, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 265; 
 (a3) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 266, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 267; 
 (a4) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 268, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 269; 
 (a5) an antibody variable region that binds to the same epitope of any of the antibody variable region selected from (a1) to (a4); and 
 (a6) an antibody variable fragment that competes with the binding of any of the antibody variable fragment selected from (a1) to (a4). 
 
     
     
         12 . The multispecific antigen-binding molecule of any one of  claims 1  to  11 , further comprises a Fc domain. 
     
     
         13 . The multispecific antigen-binding molecule of any one of  claims 1  to  12 , comprising five polypeptide chains in any one of the combination selected from (a1) to (a6) below:
 (a1) a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 201 (chain 1), a polypeptide chain comprising an amino acid sequence of SEQ ID NO: 206 (chain 2), a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 208 (chain 3) and two polypeptide chains each comprising an amino acid sequence of SEQ ID NOs: 214 (chain 4 & chain 5); 
 (a2) a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 203 (chain 1), a polypeptide chain comprising an amino acid sequence of SEQ ID NO: 206 (chain 2), a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 209 (chain 3) and two polypeptide chains each comprising an amino acid sequence of SEQ ID NOs: 214 (chain 4 & chain 5); 
 (a3) a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 204 (chain 1), a polypeptide chain comprising an amino acid sequence of SEQ ID NO: 206 (chain 2), a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 209 (chain 3) and two polypeptide chains each comprising an amino acid sequence of SEQ ID NOs: 214 (chain 4 & chain 5); 
 (a4) a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 205 (chain 1), a polypeptide chain comprising an amino acid sequence of SEQ ID NO: 206 (chain 2), a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 209 (chain 3) and two polypeptide chains each comprising an amino acid sequence of SEQ ID NOs: 214 (chain 4 & chain 5); 
 (a5) a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 216 (chain 1), a polypeptide chain comprising an amino acid sequence of SEQ ID NO: 206 (chain 2), a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 229 (chain 3) and two polypeptide chains each comprising an amino acid sequence of SEQ ID NOs: 214 (chain 4 & chain 5); 
 (a6) a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 217 (chain 1), a polypeptide chain comprising an amino acid sequence of SEQ ID NO: 206 (chain 2), a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 210 (chain 3) and two polypeptide chains each comprising an amino acid sequence of SEQ ID NOs: 214 (chain 4 & chain 5); 
 (a7) a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 219 (chain 1), a polypeptide chain comprising an amino acid sequence of SEQ ID NO: 206 (chain 2), a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 211 (chain 3) and two polypeptide chains each comprising an amino acid sequence of SEQ ID NOs: 214 (chain 4 & chain 5); 
 (a8) a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 220 (chain 1), a polypeptide chain comprising an amino acid sequence of SEQ ID NO: 206 (chain 2), a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 211 (chain 3) and two polypeptide chains each comprising an amino acid sequence of SEQ ID NOs: 214 (chain 4 & chain 5); 
 (a9) a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 221 (chain 1), a polypeptide chain comprising an amino acid sequence of SEQ ID NO: 206 (chain 2), a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 211 (chain 3) and two polypeptide chains each comprising an amino acid sequence of SEQ ID NOs: 214 (chain 4 & chain 5); 
 (a10) a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 222 (chain 1), a polypeptide chain comprising an amino acid sequence of SEQ ID NO: 206 (chain 2), a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 230 (chain 3) and two polypeptide chains each comprising an amino acid sequence of SEQ ID NOs: 214 (chain 4 & chain 5); 
 (a11) a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 223 (chain 1), a polypeptide chain comprising an amino acid sequence of SEQ ID NO: 206 (chain 2), a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 212 (chain 3) and two polypeptide chains each comprising an amino acid sequence of SEQ ID NOs: 215 (chain 4 & chain 5); 
 (a12) a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 225 (chain 1), a polypeptide chain comprising an amino acid sequence of SEQ ID NO: 206 (chain 2), a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 213 (chain 3) and two polypeptide chains each comprising an amino acid sequence of SEQ ID NOs: 215 (chain 4 & chain 5); 
 (a13) a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 226 (chain 1), a polypeptide chain comprising an amino acid sequence of SEQ ID NO: 206 (chain 2), a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 213 (chain 3) and two polypeptide chains each comprising an amino acid sequence of SEQ ID NOs: 215 (chain 4 & chain 5); 
 (a14) a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 227 (chain 1), a polypeptide chain comprising an amino acid sequence of SEQ ID NO: 206 (chain 2), a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 213 (chain 3) and two polypeptide chains each comprising an amino acid sequence of SEQ ID NOs: 215 (chain 4 & chain 5); and 
 (a15) a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 228 (chain 1), a polypeptide chain comprising an amino acid sequence of SEQ ID NO: 206 (chain 2), a polypeptide chain comprising an amino acid sequence of SEQ ID NOs: 231 (chain 3) and two polypeptide chains each comprising an amino acid sequence of SEQ ID NOs: 215 (chain 4 & chain 5). 
 
     
     
         14 . An isolated polynucleotide or plurality of polynucleotides encoding the multispecific antigen-binding molecule of any one of  claims 1  to  13 . 
     
     
         15 . A vector encoding the polynucleotide or plurality of polynucleotides of  claim 14 . 
     
     
         16 . A host cell comprising the polynucleotide or plurality of polynucleotides of  claim 14 , or the vector of  claim 15 . 
     
     
         17 . A method of producing the multispecific antigen-binding molecule of any one of  claims 1  to  13 , comprising the steps of a) culturing the host cell of  claim 16  under conditions suitable for the expression of the antigen-binding molecule and b) recovering the antigen-binding molecule. 
     
     
         18 . A pharmaceutical composition comprising the multispecific antigen-binding molecule of any one of  claims 1  to  13  and a pharmaceutically acceptable carrier. 
     
     
         19 . The multispecific antigen-binding molecule of any one of  claims 1  to  13  or the pharmaceutical composition of  claim 18 , which induces cytotoxicity, preferably T-cell-dependent cytotoxicity. 
     
     
         20 . The multispecific antigen-binding molecule of any one of  claims 1  to  13  or the pharmaceutical composition of  claim 18 , for use as a medicament. 
     
     
         21 . The multispecific antigen-binding molecule of any one of  claims 1  to  13  or the pharmaceutical composition of  claim 18 , for use in the treatment/prevention of cancer, preferably DLL3-expressing cancer or DLL3-positive cancer. 
     
     
         22 . The multispecific antigen-binding molecule or the pharmaceutical composition for use in the treatment/prevention of  claim 21 , wherein the cancer is lung cancer (preferably small cell lung cancer), or melanoma.