MULTISPECIFIC STACKED VARIABLE DOMAIN BINDING PROTEINS

1 . A multi-specific Stacked Variable Domain (SVD) binding protein comprising a tandem product of a first heavy chain variable domain sequence conjugated to a second surrogate light chain sequence, associated with a first surrogate light chain sequence conjugated to a second heavy chain variable domain sequence, wherein the tandem product comprises a first binding domain and a second binding domain, wherein each of said first and second binding domains is formed by a surrogate light chain sequence and an antibody variable domain sequence, and wherein each of said first and second binding domains binds specifically to a different binding target.

2 . The multi-specific SVD binding protein of claim 1 , wherein said first and said second binding domain are present in a single polypeptide chain.

3 . The multi-specific SVD binding protein of claim 1 , wherein said first and said second binding domain are present on more than one polypeptide chain.

4 . The multi-specific SVD binding protein of claim 1 , wherein the C-terminus of said first heavy chain variable domain sequence is conjugated to the N-terminus of said second surrogate light chain sequence.

5 . The multi-specific SVD binding protein of claim 1 , wherein the C-terminus of said first surrogate light chain sequence is conjugated to the N-terminus of said second heavy chain variable domain sequence.

6 . The multi-specific SVD binding protein of claim 1 , wherein said first heavy chain variable domain sequence and said first surrogate light chain sequence together form a first binding domain specifically binding to a first target.

7 . The multi-specific SVD binding protein of claim 1 , wherein said second surrogate light chain sequence and said second heavy chain variable domain sequence together form a second binding domain specifically binding to a second target.

8 . The multi-specific SVD binding protein of claim 1 , wherein said first and said second surrogate light chain sequences are identical.

9 . The multi-specific SVD binding protein of claim 1 , wherein said first and said second surrogate light chain sequences are different.

10 . The multi-specific SVD binding protein of claim 1 wherein said first and said second surrogate light chain sequences comprise a VpreB sequence.

11 . The multi-specific SVD binding protein of claim 10 wherein said second surrogate light chain sequence further comprises a λ5 sequence.

12 . The multi-specific SVD binding protein of claim 1 , wherein said second heavy chain variable domain sequence further comprises a heavy chain constant domain sequence.

13 . The multi-specific SVD binding protein of claim 12 , wherein said second heavy chain variable domain sequence further comprises a CH1 sequence.

14 . The multi-specific SVD binding protein of claim 12 , wherein said second heavy chain variable domain sequence further comprises an Fc region.

15 . (canceled)

16 . The multi-specific SVD binding protein of claim 1 , wherein the conjugation is by a linker sequence.

17 . (canceled)

18 . The multi-specific SVD binding protein of claim 1 , wherein the conjugation is direct fusion.

19 . The multi-specific SVD binding protein of claim 16 , wherein the linker sequence comprises a sequence selected from the group consisting of: an antibody J region sequence, a λ5 sequence, a λ light chain constant region sequence, a κ light chain constant region sequence, synthetic sequence, and any combination thereof.

20 . The multi-specific SVD binding protein of claim 19 , wherein the synthetic sequence is (Gly-Gly-Gly-Ser) n (SEQ ID NO: 109), (Gly-Gly-Gly-Gly-Ser) n (SEQ ID NO: 110), or Gly-Ala, wherein n is at least 1.

21 . The multi-specific SVD binding protein of claim 18 , wherein the C-terminus of the first heavy chain variable domain sequence is fused to the N-terminus of the second surrogate light chain sequence forming a first polypeptide chain.

22 . The multi-specific SVD binding protein of claim 18 , wherein the C-terminus of the second surrogate light chain sequence is fused to the N-terminus of the second heavy chain variable domain sequence forming a second polypeptide chain.

23 . The multi-specific SVD binding protein of claim 21 or 22 , wherein a binding site to a target is formed between a surrogate light chain sequence and a heavy chain variable domain sequence on different polypeptide chains.

24 . The multi-specific SVD binding protein of claim 21 or 22 , wherein a binding site to a target is formed between a surrogate light chain sequence and a heavy chain variable domain sequence on the same polypeptide chains.

25 . (canceled)

26 . A first polypeptide chain comprising an antibody heavy chain variable region sequence, specific for a first target, C-terminally conjugated to a polypeptide sequence comprising a VpreB sequence.

27 . The polypeptide chain of claim 26 associated with a second polypeptide chain comprising a VpreB sequence, conjugated to the N-terminus of an antibody heavy chain comprising a variable region sequence specific for a second target.

28 . The polypeptide chain of claim 27 , wherein the antibody heavy chain variable region sequence of the first polypeptide chain and the VpreB sequence of the second polypeptide chain form a binding site for said first target.

29 . A heteromeric bispecific binding protein comprising the first polypeptide chain of claim 26 , associated with the second polypeptide of claim 27 .

30 . The heteromeric bispecific binding protein of claim 29 , wherein the heavy chain variable region of the second antibody heavy chain variable region sequence specific for said second target and the VpreB sequence of the first polypeptide chain form a binding site for a second target.

31 . A heteromeric bispecific binding protein comprising one pair of the first polypeptide chain of claim 1 and one pair of the second polypeptide chain of claim 2 .

32 . The heteromeric bispecific binding protein of claim 31 , wherein the heavy chain variable region of the second antibody heavy chain variable region sequence specific for said second target and the VpreB sequence of the first polypeptide chain form a binding site for a second target.

33 - 35 . (canceled)

36 . The polypeptide chain of claim 26 or 27 or the heteromeric bispecific binding protein of claim 4 or 5 , wherein in the second polypeptide chain the conjugation is by a linker sequence.

37 - 38 . (canceled)

39 . The polypeptide chain of claim 36 , wherein the linker sequence between the antibody heavy chain variable region sequence and the VpreB sequence of the first polypeptide chain comprises a sequence selected from the group consisting of: an antibody J region sequence, an antibody constant domain region sequence, a synthetic sequence, and any combination thereof.

40 . The polypeptide chain of claim 36 , wherein the linker sequence between the antibody heavy chain variable region sequence and the VpreB sequence of the first polypeptide chain comprises a sequence selected from the group consisting of:

(SEQ ID NO:)

Xaa g Ala Ser  Xaa h ,

(SEQ ID NO:)

Xaa g Ala Ser Thr  Xaa h ,

(SEQ ID NO:)

Xaa g Ala Ser Thr Lys  Xaa h ,

(SEQ ID NO:)

Xaa g Ala Ser Thr Lys Gly  Xaa h ,

(SEQ ID NO:)

Xaa g Ala Ser Thr Lys Gly Pro  Xaa h ,

(SEQ ID NO:)

Xaa g Ala Ser Thr Lys Gly Pro Ser  Xaa h ,

(SEQ ID NO:)

Xaa g Ala Ser Thr Lys Gly Pro Ser Val  Xaa h ,

(SEQ ID NO:)

Xaa g Ala Ser Thr Lys Gly Pro Ser Val Phe  Xaa h ,

and

(SEQ ID NO:)

Xaa g Ala Ser Thr Lys Gly Pro Ser Val Phe Pro  Xaa h ,

wherein the Xaa is any amino acid, g is 0 to 10 amino acids, and h is 0 to 10 amino acids.

41 . The polypeptide chain of claim 40 , wherein Xaa g comprises a sequence selected from the group consisting of

Ser,

Ser Ser,

Val Ser Ser,

(SEQ ID NO:)

Thr Val Ser Ser,

(SEQ ID NO:)

Val Thr Val Ser Ser,

(SEQ ID NO:)

Leu Val Thr Val Ser Ser,

(SEQ ID NO:)

Thr Leu Val Thr Val Ser Ser,

(SEQ ID NO:)

Gly Thr Leu Val Thr Val Ser Ser,

(SEQ ID NO:)

Gln Gly Thr Leu Val Thr Val Ser Ser,

and

(SEQ ID NO:)

Gly Gln Gly Thr Leu Val Thr Val Ser Ser.

42 . The polypeptide chain of claim 40 , wherein Xaa h comprises a sequence selected from the group consisting of

Gln,

Gln Pro,

Gln Pro Val,

Gln Pro Val Leu,

(SEQ ID NO)

Gln Pro Val Leu His,

(SEQ ID NO:)

Gln Pro Val Leu His Gln,

(SEQ ID NO:)

Gln Pro Val Leu His Gln Pro,

(SEQ ID NO:)

Gln Pro Val Leu His Gln Pro Pro,

(SEQ ID NO:)

Gln Pro Val Leu His Gln Pro Pro Ala,

and

(SEQ ID NO:)

Gln Pro Val Leu His Gln Pro Pro Ala Met.

43 . The polypeptide chain of claim 36 , wherein the linker sequence between the antibody heavy chain variable region sequence and the VpreB sequence of the second polypeptide chain comprises a sequence selected from the group consisting of: a λ5 sequence, an antibody J region sequence, a λ light chain constant region sequence, a κ light chain constant region sequence, a synthetic sequence, and any combination thereof.

44 . The polypeptide chain of claim 36 , wherein the linker sequence between the antibody heavy chain variable region sequence and the VpreB sequence of the second polypeptide chain comprises a sequence selected from the group consisting of:

(SEQ ID NO:)

Xaa j Ser Gln  Xaa k ,

(SEQ ID NO:)

Xaa j Ser Gln Pro  Xaa k ,

(SEQ ID NO:)

Xaa j Ser Gln Pro Lys  Xaa k ,

(SEQ ID NO:)

Xaa j Ser Gln Pro Lys Ala  Xaa k ,

(SEQ ID NO:)

Xaa j Ser Gln Pro Lys Ala Thr  Xaa k ,

(SEQ ID NO:)

Xaa j Ser Gln Pro Lys Ala Thr Pro  Xaa k ,

(SEQ ID NO:)

Xaa j Ser Gln Pro Lys Ala Thr Pro Ser  Xaa k ,

(SEQ ID NO:)

Xaa j Ser Gln Pro Lys Ala Thr Pro Ser Val  Xaa k ,

(SEQ ID NO:)

Xaa j Ser Gln Pro Lys Ala Thr Pro Ser Val Thr  Xaa k ,

and

(SEQ ID NO:)

Xaa j Ser Gln Pro Lys Ala Thr Pro Ser Val Thr Gly

Gly Gly Gly Ser  Xaa k ,

wherein Xaa is any amino acid, j is 0 to 10 amino acids, and k is 0 to 6 amino acids.

45 . The polypeptide chain of claim 44 , wherein Xaa j comprises a sequence selected from the group consisting of

Leu,

Val Leu,

Thr Val Leu,

Leu Thr Val Leu,

(SEQ ID NO:)

Gln Leu Thr Val Leu,

(SEQ ID NO:)

Thr Gln Leu Thr Val Leu,

(SEQ ID NO:)

Gly Thr Gln Leu Thr Val Leu,

(SEQ ID NO:)

Ser Gly Thr Gln Leu Thr Val Leu,

(SEQ ID NO:)

and

Gly Ser Gly Thr Gln Leu Thr Val Leu.

(SEQ ID NO:)

46 . The polypeptide chain of claim 44 , wherein Xaa k comprises a sequence selected from the group consisting of

Gln,

Gln Val,

Gln Val Gln,

Gln Val Gln Leu,

(SEQ ID NO:)

Gln Val Gln Leu Val,

(SEQ ID NO:)

and

Gln Val Gln Leu Val Gln.

(SEQ ID NO:)

47 . (canceled)

48 . The polypeptide chain of claim 26 or 27 , or the heteromeric bispecific binding protein of claim 4 or 5 , wherein the VpreB sequence is fused, at its C-terminus, to a heterologous sequence.

49 . The polypeptide chain or the heteromeric bispecific binding protein of claim 48 , wherein the heterogenous sequence is selected from the group consisting of a λ5 sequence, an antibody J-region sequence, and a light chain constant domain region sequence.

50 . A first polypeptide chain comprising an antibody heavy chain variable region sequence specific for a first target, C-terminally conjugated to a first polypeptide sequence comprising a first VpreB sequence, wherein the first polypeptide sequence comprising the VpreB sequence is C-terminally conjugated to a second polypeptide sequence comprising a second VpreB sequence, conjugated to a heterologous sequence.

51 . The polypeptide chain of claim 50 associated with a second polypeptide chain comprising an antibody heavy chain comprising a variable region sequence specific for a second polypeptide target.

52 . The polypeptide chain of claim 50 , wherein the antibody heavy chain variable region sequence of the first polypeptide chain and the first VpreB sequence of the first polypeptide chain form a binding site for said first target.

53 . A heteromeric bispecific binding protein comprising one pair of the first polypeptide chain of claim 50 and one pair of the second polypeptide chain of claim 51 .

54 . The heteromeric bispecific binding protein of claim 53 , wherein the heavy chain variable region of the second antibody heavy chain variable region sequence specific for said second target and the second VpreB sequence of the first polypeptide chain form a binding site for a second target.

55 . The polypeptide chain of claim 50 or 51 or the heteromeric bispecific binding protein of claim 52 or 53 , wherein in the first polypeptide chain the conjugation is by a linker sequence.

56 - 57 . (canceled)

58 . The polypeptide chain of claim 55 , wherein the linker sequence between the antibody heavy chain variable region sequence and the first polypeptide sequence comprising a first VpreB sequence comprises the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 108).

59 . The polypeptide chain of claim 55 , wherein the linker sequence between the first polypeptide sequence comprising a first VpreB sequence and the second polypeptide sequence comprising a second VpreB sequence comprises the amino acid sequence Gly-Ala.

60 - 62 . (canceled)

63 . A polypeptide chain comprising an antibody heavy chain variable region sequence specific for a first target, C-terminally conjugated to a first polypeptide sequence comprising a first surrogate light chain (SLC) sequence, wherein the first SLC sequence is C-terminally conjugated to an antibody heavy chain variable region sequence specific for a second target.

64 - 75 . (canceled)

76 . Original) A first polypeptide chain comprising an antibody heavy chain variable region sequence specific for a first target conjugated to a first polypeptide sequence comprising a first VpreB sequence, wherein the first polypeptide sequence comprising the first VpreB sequence is C-terminally conjugated to a second polypeptide sequence comprising a dimerization domain.

77 . The polypeptide chain of claim 76 associated with a second polypeptide chain comprising a first polypeptide sequence that comprises a second VpreB sequence, wherein the first polypeptide sequence comprising the second VpreB sequence is C-terminally conjugated to an antibody heavy chain variable region sequence specific for a second target.

78 - 81 . (canceled)

82 . A heteromeric bispecific binding protein comprising the first and second polypeptide chains of claim 77 , associated with each other.

83 . The heteromeric bispecific binding protein of claim 82 , wherein the heavy chain variable region sequence specific for said second target of the second polypeptide and the first VpreB sequence of the first polypeptide chain form a binding site for a second target.

84 . The polypeptide chain of claim 76 or heteromeric bispecific binding protein of claim 83 , wherein the conjugation is by a linker sequence.

85 - 86 . (canceled)

87 . The polypeptide chain or heteromultimeric bispecific binding protein of claim 84 , wherein the linker sequence comprises a sequence selected from the group consisting of: an antibody J region sequence, a λ5 sequence, a λ light chain constant region sequence, a κ light chain constant region sequence, synthetic sequence, and any combination thereof.

88 . (canceled)

89 . The polypeptide chain of claim 76 or 77 , or the heteromeric bispecific binding protein of claim 82 or 83 , wherein the VpreB sequence is fused, at its C-terminus, to a heterologous sequence.

90 . The polypeptide chain or the heteromeric bispecific binding protein of claim 89 , wherein the heterogenous sequence is selected from the group consisting of a λ5 sequence and a light chain constant domain region sequence.

91 . The polypeptide chain of claim 76 , wherein one or both of the dimerization domains comprise an engineered amino acid sequence that promotes interaction between the dimerization domains.

92 . The polypeptide chain of claim 91 , wherein the engineered amino acid sequence comprises a region selected from the group consisting of: a complementary hydrophobic region, a complementary hydrophilic region, and a compatible protein-protein interaction domain.

93 . A first polypeptide chain comprising an antibody heavy chain variable region sequence specific for a first target C terminally conjugated to a first polypeptide sequence comprising a first VpreB sequence, wherein the N-terminus of the antibody heavy chain variable region sequence specific for a first target is conjugated to a dimerization domain.

94 . The polypeptide chain of claim 93 associated with a second polypeptide chain comprising a first polypeptide sequence that comprises a second VpreB sequence, wherein the C-terminus of the first polypeptide sequence comprising the second VpreB sequence is conjugated to an antibody heavy chain variable region sequence specific for a second target and the N-terminus of the first polypeptide sequence comprising the second VpreB sequence is conjugated to a dimerization domain.

95 - 97 . (canceled)

98 . A heteromeric bispecific binding protein comprising the first and second polypeptide chains of claim 94 , associated with each other.

99 - 108 . (canceled)

109 . A heteromeric trispecific binding protein comprising a first polypeptide chain comprising an antibody heavy chain variable region sequence specific for a first target, C-terminally conjugated to a polypeptide sequence comprising a first VpreB sequence, wherein the first polypeptide chain is associated with

a) a second polypeptide chain comprising a polypeptide sequence that comprises a second VpreB sequence conjugated to the N-terminus of an antibody heavy chain comprising a variable region sequence specific for a second target; and

b) a third polypeptide chain comprising a polypeptide sequence that comprises a third VpreB sequence conjugated to the N-terminus of an antibody heavy chain comprising a variable region sequence specific for a third target.

110 - 127 . (canceled)