Antigen-binding molecule containing modified antibody variable region

1. A method of selecting an antigen-binding molecule that recognizes a first antigen that is human CD3 and a second antigen different from the first antigen, the method comprising:

identifying a starting antigen-binding molecule comprising a starting antibody variable region comprising a VH and a VL that together recognize the first antigen and do not recognize the second antigen;

preparing a library of altered antigen-binding molecules comprising variable regions that vary from the starting antibody variable region and from each other by at least one amino acid substitution or insertion at a position or positions located within any one or more of a VH CDR1, a VH CDR2, a VH CDR3, a VH FR3 region, a VL CDR1, a VL CDR2, or a VL CDR3; and

selecting from the library an altered antigen-binding molecule comprising an altered antibody variable region comprising a first antigen-binding site and a second antigen-binding site, wherein:

(a) the first antigen-binding site recognizes the first antigen; and

(b) the second antigen-binding site recognizes the second antigen, wherein the second antigen is selected from an Fcγ receptor (FcγR), a toll-like receptor (TLR), a lectin, an IgA, an immune checkpoint molecule, a tumor necrosis factor (TNF) superfamily molecule, a TNF receptor (TNFR) superfamily molecule, and an NK receptor molecule; and

(c) the first antigen-binding site cannot bind to the first antigen when the second antigen-binding site is bound to the second antigen; and

(d) the second antigen-binding site cannot bind to the second antigen when the first antigen-binding site is bound to the first antigen,

thereby selecting an antigen-binding molecule that recognizes the first antigen and the second antigen.

2. The method according to claim 1 , wherein the first antigen is naturally expressed on a first type of cell and the second antigen is naturally expressed on a different type of cell.

3. The method according to claim 1 , wherein at least one of the amino acid substitutions or insertions in the library of altered antigen-binding molecules is an insertion of 1 to 25 amino acids.

4. The method according to claim 1 , wherein the altered antibody variable region comprises at least one amino acid substitution or insertion at a position located within a VH CDR1, a VH CDR2, a VH CDR3, or a VH FR3 region of the altered antibody variable region.

5. The method according to claim 1 , wherein the altered antibody variable region comprises at least one amino acid substitution or insertion at a position located within a loop of the altered antibody variable region.

6. The method according to claim 1 , wherein at least one of the amino acid substitutions or insertions is at a position selected from heavy chain variable domain Kabat numbering positions 31 to 35, 50 to 65, 71 to 74, and 95 to 102, and light chain variable domain Kabat numbering positions 24 to 34, 50 to 56, and 89 to 97.

7. A method of selecting an antigen-binding molecule that recognizes a first antigen that is human CD3 and a second antigen that is not human CD3, the method comprising:

identifying a starting antigen-binding molecule comprising a starting antibody variable region comprising a VH and a VL that together recognize the first antigen and do not recognize the second antigen;

preparing a library of altered antigen-binding molecules comprising variable regions that vary from the starting antibody variable region and from each other by at least one amino acid substitution or insertion at a position or positions located within a VH CDR1, a VH CDR2, a VH CDR3, a VH FR3 region, a VL CDR1, a VL CDR2, or a VL CDR3; and

selecting from the library an altered antigen-binding molecule comprising an altered antibody variable region comprising a first antigen-binding site and a second antigen-binding site, wherein:

(a) the first antigen-binding site recognizes the first antigen, and

(b) the second antigen-binding site recognizes the second antigen and does not recognize human CD3, and

(c) the first antigen-binding site cannot bind to the first antigen when the second antigen-binding site is bound to the second antigen, and

(d) the second antigen-binding site cannot bind to the second antigen when the first antigen-binding site is bound to the first antigen,

thereby selecting an antigen-binding molecule that recognizes the first antigen and the second antigen.

8. The method according to claim 7 , wherein the second antigen is naturally expressed on a cell that is not a T cell.

9. The method according to claim 7 , wherein the second antigen-binding site is located within a loop of the first antigen-binding site.

10. The method according to claim 7 , wherein the second antigen-binding site is located within a VH CDR3 or VL CDR3 of the first antigen-binding site.

11. The method according to claim 7 , wherein the second antigen is a molecule expressed on the surface of a T cell or any other immunocyte.

12. The method according to claim 7 , wherein the second antigen-binding site is a peptide.

13. The method according to claim 12 , wherein the peptide binds to an antigen selected from a vascular endothelial growth factor receptor (VEGFR), a tumor necrosis factor receptor (TNFR), a toll-like receptor (TLR) 5, a TLR4, a TLR2, a T cell very late antigen (VLA) receptor, a platelet-derived growth factor receptor (PDGFR), a Naip5 Nod-like receptor (NLR), an integrin, FcγRIIa, an epidermal growth factor receptor (EGFR), a death receptor (DR) 5 agonist, a C—X-C chemokine receptor type 4 (CXCR4), a cluster of differentiation (CD) 40, and a CD154.

14. The method according to claim 7 , wherein the second antigen-binding site is located in the altered antibody variable region, at a position selected from heavy chain variable domain Kabat numbering positions 31 to 35, 50 to 65, 71 to 74, and 95 to 102, and light chain variable domain Kabat numbering positions 24 to 34, 50 to 56, and 89 to 97.

15. The method of claim 7 , wherein the altered antibody variable region comprises a heavy chain variable domain amino acid sequence comprising, at each of the following Kabat numbering positions, one of the amino acid residues indicated for that position:

31: Ile, Asn, or Ser;

32: Ala;

33: Trp;

34: Met;

35: His;

50: Gln;

51: Ile;

52: Lys;

52(a): Asp;

52(b): Arg or Lys;

52(c): Ala, Gly, Leu, Gln, Val, or Ser;

53: Gln, Ser, or Asn;

54: Ala, Gly, Leu, Gln, Ser, or Asn;

55: Tyr;

56: Leu, Asn, or Ala;

57: Ala, Asn, or Thr;

58: Tyr;

59: Tyr;

60: Ala;

61: Pro or Glu;

62: Ser;

63: Val;

64: Lys;

65: Gly;

95: Val;

96: His;

97: Tyr;

98: Ala, Gly, Pro, Ser, Thr, Gln, or His;

99: Ala, Leu, Ser, Gln, or Thr;

100: Ala, Val, Gly, Ser, Thr, Tyr, or Phe;

100(a): Ala, Val, Gly, Ser, Thr, Tyr, Phe, or Asp;

100(b): Gly, Ser, Thr, Tyr, Phe, or Asp;

100(c): Ala, Val, Leu, Gly, Ser, Tyr, or Phe;

100(d): Leu, Gly, Ser, Tyr, or Phe;

100(e): Pro, Gly, Ser, Tyr, or Phe;

100(f): Ala, Ser, Gln, Lys, Tyr, or Gly;

100(g): Tyr, Phe, or Gly;

100(h): Gly;

100(i): Val;

101: Asp;

102: Ile.

16. The method of claim 7 , wherein the altered antibody variable region comprises a light chain variable domain amino acid sequence comprising, at each of the following Kabat numbering positions, one of the amino acid residues indicated for that position:

24: Arg, Ala, Glu, Gly, Gln, Ser, Thr, or Tyr;

25: Ser Ala, Pro, or Thr;

26: Ser or Asp;

27: Gln, Asp, or Glu;

27(a): Ser, Glu, or Pro;

27(b): Leu, Ile, Pro, or Val;

27(c): Val or Leu;

27(d): His;

27(e): Ser, Ala, Gly, Ile, Leu, Met, Asn, Pro, Gln, Thr, or Val;

28: Asn;

29: Arg;

30: Asn, Phe, His, Met, Gln, or Tyr;

31: Thr or Ile;

32: Tyr;

33: Leu, Ala, Ile, or Val;

34: His, Ala, Gly, Thr, or Val;

45: Gln;

50: Lys;

51: Val, Gly, or Ile;

52: Ser, Ala, Gln, or Tyr;

53: Asn, Pro, or Trp;

54: Arg or Pro;

55: Phe, Ala, Gly, His, Lys, Asn, Pro, or Tyr;

56: Ser, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Thr, Val, or Tyr;

74: Lys or Thr;

77: Arg or Ser;

89: Gly, Ala, Asn, or Thr;

90: Gln or Glu;

91: Gly;

92: Thr or Ser;

93: Gln, Ala, or Ser;

94: Val, Ala, Asp, Asn, Ser, or Thr;

95: Pro;

96: Tyr or Phe;

97: Thr;

107: Lys or Glu.

17. The method according to claim 7 , wherein the second antigen-binding site comprises an amino acid residue or a peptide that is inserted in the altered antibody variable region between a pair of positions selected from the following pairs of Kabat numbering positions:

heavy chain variable domain positions 52b and 52c,

heavy chain variable domain positions 72 and 73,

heavy chain variable domain positions 98 and 99,

heavy chain variable domain positions 100 and 100a.

18. The method according to claim 1 , wherein the second antigen is a TNFR superfamily molecule.