Phage displaying system expressing single chain antibody

Abstract

Disclosed are nucleic acid libraries for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, and for facilitating production of a disulfide-stabilized single chain antibody. Also disclosed are host cell libraries and phage libraries including the nucleic acid libraries. Further disclosed are methods for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, and methods for producing a disulfide-stabilized single chain antibody and non-fusion form thereof.

1 . A nucleic acid library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, the library comprising a plurality of expression constructs, each of which includes
 a first nucleotide sequence encoding a signal peptide, and   a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3′ downstream to the first nucleotide, wherein the signal peptide has the amino acid sequence of:   (a) VKKLLX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 0 X 10 AAQPAMAHHHHHHGH (SEQ ID NO:1),   (b) VKKLLFAIPLX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 MAHHHHHHGH (SEQ ID NO:2), or   (c) VKKLLFAIPLVVPFYX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 HHHGH (SEQ ID NO:3),   each of X 1 -X 10  in (a), (b), and (c) being one of the 20 naturally occurring amino acid residues.   
     
     
         2 . The nucleic acid library of  claim 1 , wherein each of the expression constructs further includes a third nucleotide encoding a phage coat protein, the third nucleotide sequence being located 3′ downstream to the second nucleotide sequence. 
     
     
         3 . The nucleic acid library of  claim 2 , wherein the expression construct is a phagemid. 
     
     
         4 . The nucleic acid library of  claim 1 , wherein the single chain antibody contains a first variable region, a second variable region, and a protein linker connecting the first and the second variable region, wherein the first and the second variable region are stabilized by an interface disulfide bond. 
     
     
         5 . The nucleic acid library of  claim 4 , wherein the first variable region is a heavy chain variable region (V H ) or a light chain variable region (V L ). 
     
     
         6 . The nucleic acid library of  claim 5 , wherein the second variable region is a heavy chain variable region (V H ) or a light chain variable region (V L ). 
     
     
         7 . A host cell library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, the library comprising a plurality of host cells each containing an expression construct that includes
 a first nucleotide sequence encoding a signal peptide, and   a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3′ downstream to the first nucleotide, wherein the signal peptide has the amino acid sequence of:   (a) VKKLLX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 AAQPAMAHHHHHHGH (SEQ ID NO:1),   (b) VKKLLFAIPLX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 MAHHHHHHGH (SEQ ID NO:2), or   (c) VKKLLFAIPLVVPFYX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 HHHGH (SEQ ID NO:3),   each of X 1 -X 10  in (a), (b), and (c) being one of the 20 naturally occurring amino acid residues.   
     
     
         8 . The host cell library of  claim 6 , wherein each of the expression constructs further includes a third nucleotide encoding a phage coat protein, the third nucleotide sequence being located 3′ downstream to the second nucleotide sequence. 
     
     
         9 . A phage library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, the library comprising a plurality of phage particles each containing a disulfide-stabilized single chain antibody fused with a coat protein on the surface of said phage, wherein the phage library is prepared by the steps of:
 providing a host cell containing an expression construct, and   culturing the host cell in a medium under conditions allowing expression of the plurality of phage particles,   
       wherein the expression construct that includes
 a first nucleotide sequence encoding a signal peptide, 
 a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3′ downstream to the first nucleotide, and 
 a third nucleotide sequence encoding a phage envelop protein, the third nucleotide sequence being located 3′ downstream to the second nucleotide sequence, 
 
       wherein the signal peptide has the amino acid sequence of
 (a) VKKLLX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 AAQPAMAHHHHHHGH (SEQ ID NO:1), 
 (b) VKKLLFAIPLX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 MAHHHHHHGH (SEQ ID NO:2), or 
 (c) VKKLLFAIPLVVPFYX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 HHHGH (SEQ ID NO:3), 
 each of X 1 -X 10  in (a), (b), and (c) being one of the 20 naturally occurring amino acid residues. 
 
     
     
         10 . An isolated nucleic acid, comprising
 a first nucleotide sequence encoding a signal peptide, and   a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3′ downstream to the first nucleotide, wherein the signal peptide has the amino acid sequence of:   (a) VKKLLX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 AAQPAMAHHHHHHGH (SEQ ID NO:596), in which X 1  is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X 2  is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X 3  is A, F, G, L, M, P, Q, R, S, T, V, or W; X 4  is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X 5  is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X 6  is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X 7  is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X 8  is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X 9  is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X 10  is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;   (b) VKKLLFAIPLX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 MAHHHHHHGH (SEQ ID NO:597), in which X 1  A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X 2  is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X 3  is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X 4  is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X 5  is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X 6  is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X 7  is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X 8  is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X 9  is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X 10  is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or   (c) VKKLLFAIPLVVPFYX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 HHHGH (SEQ ID NO:598), in which X 1  is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X 2  is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X 3  is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X 4  is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X 5  is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X 6  is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X 7  is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X 8  is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X 9  is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X 10  is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.   
     
     
         11 . The nucleic acid of  claim 10 , further comprising a third nucleotide encoding a phage coat protein, the third nucleotide sequence being located 3′ downstream to the second nucleotide sequence. 
     
     
         12 . The nucleic acid of  claim 10 , wherein the nucleic acid is an expression vector for expression a fusion protein containing the signal peptide and the single chain antibody. 
     
     
         13 . The nucleic acid of  claim 10 , wherein the single chain antibody contains a first variable region, a second variable region, and a protein linker connecting the first and the second variable region, wherein the first and the second variable region are stabilized by an interface disulfide bond. 
     
     
         14 . The nucleic acid library of  claim 13 , wherein the first variable region is a heavy chain variable region (V H ) or a light chain variable region (V L ). 
     
     
         15 . The nucleic acid library of  claim 13 , wherein the second variable region is a heavy chain variable region (V H ) or a light chain variable region (V L ). 
     
     
         16 . The nucleic acid of  claim 12 , wherein the expression vector is a phagemid. 
     
     
         17 . A host cell containing the nucleic acid of  claim 13 . 
     
     
         18 . A phage containing a disulfide-stabilized single chain antibody fused with its coat protein on the surface, wherein the phage is prepared by the method comprising the steps of:
 providing a host cell of  claim 17 , and   culturing the host cell in a medium under conditions allowing expression of the phage.   
     
     
         19 . A method for producing a disulfide-stabilized single chain antibody, comprising
 providing a host cell containing an expression construct, and   culturing the host cell in a medium under conditions allowing expression of the disulfide-stabilized single chain antibody,   
       wherein the expression construct includes a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3′ downstream to the first nucleotide, and 
       wherein the signal peptide has the amino acid sequence of
 (a) VKKLLX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 AAQPAMAHHHHHHGH (SEQ ID NO:596), in which X 1  is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X 2  is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X 3  is A, F, G, L, M, P, Q, R, S, T, V, or W; X 4  is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X 5  is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X 6  is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X 7  is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X 8  is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X 9  is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X 10  is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; 
 (b) VKKLLFAIPLX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 MAHHHHHHGH (SEQ ID NO:597), in which X 1  A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X 2  is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X 3  is A, C, D, F, G, H, I, L, M, N, P, O, R, S, T, V, W, or Y; X 4  is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X 5  is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X 6  is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X 7  is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X 8  is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X 9  is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X 10  is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or 
 (c) VKKLLFAIPLVVPFYX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 HHHGH (SEQ ID NO:598), in which X 1  is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X 2  is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X 3  is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X 4  is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X 5  is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X 6  is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X 7  is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X 8  is A, E, F, G, H, I, K, L, M, N, P, O, R, S, T, V, W, or Y; X 9  is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X 10  is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y. 
 
     
     
         20 . The method of  claim 19 , wherein the single chain antibody contains a first variable region, a second variable region, and a protein linker connecting the first and the second variable region, wherein the first and the second variable region are stabilized by an interface disulfide bond. 
     
     
         21 . The method of  claim 20 , wherein the first variable region is a heavy chain variable region (V H ) or a light chain variable region (V L ). 
     
     
         22 . The method of  claim 20 , wherein the second variable region is a heavy chain variable region (V H ) or a light chain variable region (V L ). 
     
     
         23 . The method of  claim 19 , further comprising, after the culturing step, collecting the medium for isolating the disulfide-stabilized single chain antibody. 
     
     
         24 . The method of  claim 19 , wherein the expression construct is a phagemid that further includes a third nucleotide encoding a phage envelope protein, the third nucleotide sequence being located 3′ downstream to the second nucleotide sequence. 
     
     
         25 . The method of  claim 24 , further comprising, after the culturing step, collecting the medium for isolating phage particles that display the disulfide-stabilized single chain antibody. 
     
     
         26 . An array of disulfide-stabilized single chain antibodies produced by the method of  claim 19  coated on a solid surface. 
     
     
         27 . The array of  claim 26 , wherein the solid surface is selected from the group consisting of silicon, plastic, nylon, glass, ceramic, photoresist, and rubber surface. 
     
     
         28 . An isolated signal peptide that facilitates production of a disulfide-stabilized single chain antibody, which is one selected from the group consisting of the peptides having the amino acid sequences set forth in SEQ ID NOS: 5-593. 
     
     
         29 . The signal peptide of  claim 28 , which is one selected from the group consisting of the peptides having the amino acid sequences set forth in SEQ ID NOS: 5-16, 18-19, 21-29, 31-36, 38-42, 45, 48-53, 55, 57-64, 255-304, 381-429 and 476. 
     
     
         30 . An isolated nucleic acid for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, or for facilitating production of a disulfide-stabilized single chain antibody, which is a nucleic acid encoding a signal peptide selected from the group consisting of the peptides having the amino acid sequences set forth in SEQ ID NOS: 5-593. 
     
     
         31 . The nucleic acid of  claim 30 , which is a nucleic acid encoding a signal peptide selected from the group consisting of the peptides having the amino acid sequences set forth in SEQ ID NOS: 5-16, 18-19, 21-29, 31-36, 38-42, 45, 48-53, 55, 57-64, 255-304, 381-429 and 476.