Compositions and methods using a single-domain antibody binding transferrin and protein A for increasing protein half-life in serum

1. An isolated antibody or antigen-binding fragment thereof that specifically binds human and cynomolgus monkey serum transferrin protein and protein A resin, wherein the isolated antibody or antigen-binding fragment thereof can be purified by a protein A column, and wherein the isolated antibody or antigen-binding fragment thereof comprises:

(a) a complementarity determining region (CDR)1 having an amino acid sequence selected from the group consisting of SEQ ID NO:46-SEQ ID NO:90;

(b) a CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:136-SEQ ID NO:180; and

(c) a CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:226-270.

2. The isolated antibody or antigen-binding fragment thereof of claim 1 , wherein the isolated antibody or antigen-binding fragment thereof is a single-domain antibody (sdAb).

3. The isolated antibody or antigen-binding fragment thereof of claim 2 , comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NO: 316-SEQ ID NO: 360.

4. The isolated antibody or antigen-binding fragment thereof of claim 3 , comprising an amino acid sequence at least 95% identical to the amino acid sequence of

AS01274

(QVQLVESGGGLVQPGGSLRLSCVASGSIASIATMAWYRQAPGQQRELVA

GITRGGSTKYADSVKGRFTISRDNAKNTLYLQMNSLKPDDTAVYYCTDYS

RKYYQDYWGQGTQVTVSS (SEQ ID NO: 316));

or

AS01299

(QVQLVESGGGLVQPGGSLRLSCAASGSIFSIATMAWYRQAPGKQRELVA

GITRSGSTNYRDSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCTDYS

SRYYHDYWGQGTQVTVSS (SEQ ID NO: 332));

or

AS01299VH3a

(EVQLVESGGGLVQPGGSLRLSCAASGSIFSIATMAWYRQAPGKGLELVA

GITRSGSTNYRDSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCTDYS

SRYYHDYWGQGTLVTVSS (SEQ ID NO: 354);

or

AS01299VH3a-A49

(EVQLVESGGGLVQPGGSLRLSCAASGSIFSIATMAWYRQAPGKGLELVS

GITRSGSTNYRDSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCTDYS

SRYYHDYWGQGTLVTVSS (SEQ ID NO: 356));

or

AS01299VH3a-L47

(EVQLVESGGGLVQPGGSLRLSCAASGSIFSIATMAWYRQAPGKGLEWVA

GITRSGSTNYRDSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCTDYS

SRYYHDYWGQGTLVTVSS (SEQ ID NO: 357));

or

AS01299VH3a-M78

(EVQLVESGGGLVQPGGSLRLSCAASGSIFSIATMAWYRQAPGKGLELVA

GITRSGSTNYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTDYS

SRYYHDYWGQGTLVTVSS (SEQ ID NO: 358));

or

AS01299VH4

(EVQLVESGGGLVQPGGSLRLSCAASGSIFSIATMAWYRQAPGKGLELVS

GITRSGSTNYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTDYS

SRYYHDYWGQGTLVTVSS (SEQ ID NO: 359));

or

AS01299VH4-L47

(EVQLVESGGGLVQPGGSLRLSCAASGSIFSIATMAWYRQAPGKGLEWVS

GITRSGSTNYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTDYS

SRYYHDYWGQGTLVTVSS (SEQ ID NO: 360)).

5. The isolated antibody or antigen-binding fragment thereof of claim 1 , having a dissociation constant (K D ) of 10 −7 M or less.

6. The isolated antibody or antigen-binding fragment thereof of claim 1 , wherein the isolated antibody or antigen-binding fragment thereof is produced recombinantly.

7. A fusion protein comprising the isolated antibody or antigen-binding fragment thereof of claim 1 , a target protein, and a linker, wherein the isolated antibody or antigen-binding fragment thereof is fused to the carboxyl-terminus or amino-terminus of the target protein, and the linker separates the antibody and the carboxyl-terminus or amino-terminus of the target protein.

8. A composition comprising the fusion protein of claim 7 and transferrin.

9. A fusion protein comprising the isolated antibody or antigen-binding fragment thereof of claim 1 , wherein the fusion protein is capable of being purified by protein A resin.

10. A method of increasing the half-life of a target protein in a fusion protein, the method comprising exposing the fusion protein of claim 7 to transferrin in cynomolgus monkey serum to thereby increase the half-life of the target protein in the fusion protein.

11. A method of producing the fusion protein of claim 7 , comprising:

(a) obtaining an expression vector encoding the fusion protein;

(b) introducing the expression vector into a cell to obtain a recombinant cell;

(c) growing the recombinant cell under conditions to allow expression of the fusion protein; and

(d) obtaining the fusion protein from the recombinant cell or its supernatant.

12. A method for increasing the half-life of a target protein, the method comprising:

(1) obtaining a fusion protein, wherein the fusion protein comprises the isolated antibody or antigen-binding fragment thereof of claim 1 , a target protein, and a linker, wherein the isolated antibody or antigen-binding fragment thereof is fused to the carboxyl-terminus or amino-terminus of the target protein, and the linker separates the isolated antibody or antigen-binding fragment thereof and the carboxyl-terminus or amino-terminus of the target protein, and

(2) exposing the fusion protein to transferrin,

wherein the transferrin increases the half-life of the target protein in the fusion protein compared to the target protein alone.

13. The method of claim 12 , wherein the exposing step comprises administering the fusion protein to a serum comprising the transferrin of cynomolgus monkey.

14. A system for increasing the half-life of a target protein, the system comprising:

(a) a first nucleotide sequence encoding the isolated antibody or antigen-binding fragment thereof of claim 1 , and a second nucleotide sequence encoding a linker, wherein the first and second nucleotide sequences are operably linked;

(b) a host cell; and

(c) transferrin.