Bi-specific monovalent diabodies that are capable of binding CD19 and CD3, and uses thereof

1. A CD19×CD3 bi-specific Fc diabody that specifically binds to CD19 and to CD3, wherein the diabody comprises a first, a second and a third polypeptide chain, wherein said polypeptide chains form a covalently bonded complex, and wherein:

I. said first polypeptide chain comprises, in the N-terminal to C-terminal direction:

A. a Domain IA, comprising

(1) a sub-Domain (IA1), which comprises a VL Domain that binds to either CD19 (VL CD19 ) or CD3 (VL CD3 ); and

(2) a sub-Domain (IA2), which comprises a VH Domain that binds to either CD19 (VH CD19 ) or CD3 (VH CD3 );

wherein said sub-Domains IA1 and IA2 are separated from one another by a polypeptide linker, and are either

(a) VL CD19 and VH CD3 ; or

(b) VL CD3 and VH CD19 ;

B. a Domain IB, comprising a positively or negatively charged helical domain, wherein said Domain IB is separated from said Domain 1A by a polypeptide linker;

C. a Domain IC, comprising a CH2-CH3 Domain of an antibody; and

II. said second polypeptide chain comprises, in the N-terminal to C-terminal direction:

A. a Domain IIA, comprising

(1) a sub-Domain (IIA1), which comprises a VL Domain that binds to either CD19 (VL CD19 ) or CD3 (VL CD3 ); and

(2) a sub-Domain (IIA2), which comprises a VH Domain that binds to either CD19 (VH CD19 ) or CD3 (VH CD3 );

wherein said sub-Domains IIA1 and IIA2 are separated from one another by a polypeptide linker, and are:

(a) VL CD19 and VH CD3 , if said sub-Domains IA1 and IA2 are VL CD3 and VH CD19 ; or

(b) VL CD3 and VH CD19 , if said sub-Domains IA1 and IA2 are VL CD19 and VH CD3 ;

B. a Domain IIB, comprising a positively or negatively charged helical domain, wherein said Domain IIB is separated from said Domain IIA by a polypeptide linker, and wherein said charged helical domain of said Domain D3 and said charged helical domain of said Domain IIB have opposite charges, wherein:

one of said charged helical domains of said Domain IB and said Domain IIB comprises the amino acid sequence of SEQ ID NO:12 and the other of said charged helical domains of said Domain IB and said Domain IIB comprises the amino acid sequence of SEQ ID NO:13; and

III. said third polypeptide chain comprises, in the N-terminal to C-terminal direction, a Domain IIIC that comprises a CH2-CH3 Domain of an antibody;

wherein:

(1) said VL CD19 comprises the amino acid sequence of SEQ ID NO:17 and said VH CD19 comprises the amino acid sequence of SEQ ID NO:21; and

(2) said VL CD3 comprises the amino acid sequence of SEQ ID NO:25 and said VH CD3 comprises the amino acid sequence of SEQ ID NO:29; and

(3) said VL CD19 and said VH CD19 domains form a monovalent CD19 binding domain; and

(4) said VL CD3 and VH CD3 domains form a monovalent CD3 binding domain; and

(5) said CH2-CH3 Domains of said first and third polypeptide chains form an Fc domain that binds to an Fc receptor, thereby forming said CD19×CD3 bi-specific Fc diabody.

2. The CD19×CD3 bi-specific Fc diabody of claim 1 , wherein:

(A) said Domains IB and IIB each comprise a cysteine residue that covalently bonds said first polypeptide chain to said second polypeptide chain via a disulfide bond; and

(B) said Domains IC and IIIC each comprise a cysteine residue that covalently bonds said first polypeptide chain to said third polypeptide chain via a disulfide bond.

3. The CD19×CD3 bi-specific Fc diabody of claim 1 , wherein said CH2-CH3 Domain of said Domain IC comprises the amino acid sequence of SEQ ID NO:15 and said CH2-CH3 Domain of said Domain IIIC comprises the amino acid sequence of SEQ ID NO:16.

4. The CD19×CD3 bi-specific Fc diabody of claim 1 , wherein:

said charged helical domain of said Domain D3 comprises the amino acid sequence of SEQ ID NO:12 and said charged helical domain of said Domain IIB comprises the amino acid sequence of SEQ ID NO:13.

5. The CD19×CD3 bi-specific Fc diabody of claim 1 , wherein:

(A) said first polypeptide chain comprises the amino acid sequence of SEQ ID NO:35;

(B) said second polypeptide chain comprises the amino acid sequence of SEQ ID NO:37; and

(C) said third polypeptide chain comprises the amino acid sequence of SEQ ID NO:39.

6. The CD19×CD3 bi-specific Fc diabody of claim 1 , which is capable of cross-reacting with both human and primate CD19 and with both human and primate CD3.

7. A pharmaceutical composition comprising the CD19×CD3 bi-specific Fc diabody of claim 1 , and a physiologically acceptable carrier.

8. A pharmaceutical composition comprising the CD19×CD3 bi-specific Fc diabody of claim 5 , and a physiologically acceptable carrier.

9. A covalently associated polypeptide complex, wherein said polypeptide complex comprises a first polypeptide chain and a second polypeptide chain, wherein:

(A) said first polypeptide chain comprises a polypeptide having the amino acid sequence of SEQ ID NO:2 linked to a charged helical domain having the amino acid sequence of SEQ ID NO:12; and

(B) said second polypeptide chain comprises a polypeptide having the amino acid sequence of SEQ ID NO:2 linked to a charged helical domain having the amino acid sequence of SEQ ID NO:13;

wherein said charged helical domain of said first polypeptide chain and said charged helical domain of said second polypeptide chain are covalently bonded to one another via a disulfide bond.