US20110082052A1

US20110082052A1 - Phage displaying system expressing single chain antibody

Info

Publication number: US20110082052A1
Application number: US12/854,632
Authority: US
Inventors: An-Suei Yang; Yu-Ching Lee; Ing-Chien Chen; Chung-Ming Yu; Hung-Ju Hsu; Yi-Jen Huang; Hung-Ju Chang; Keng-Chang Tsai; Hung-Pin Peng
Original assignee: Academia Sinica
Current assignee: Academia Sinica
Priority date: 2009-08-11
Filing date: 2010-08-11
Publication date: 2011-04-07
Also published as: US9309522B2; US9951121B2; TW201118205A; TWI506138B; US20160176952A1; WO2011019827A2; TWI449821B; WO2011019827A9; US20130323787A1; TW201500550A

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.

Description

RELATED APPLICATION

This application claims priority to U.S. provisional application 61/232,819 filed on Aug. 11, 2009. The prior application is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention is related to a phage displaying system expressing disulfide-stabilized single chain antibody variable fragments (sc-dsFv).

BACKGROUND OF THE INVENTION

A single chain variable fragment (scFv) is a single polypeptide chain antibody fragment having a light chain variable domain and a heavy chain variable domain, with a flexible linkage peptide connecting the two domains. An scFv displayed as a fusion protein N-terminal to the pIII minor capsid protein on filamentous phage surface is one of the most prominent methods in antibody engineering. It was reported that the small size of the scFv enabled superior tissue-penetrating capabilities over whole IgG or Fab fragment, making scFv an ideal scaffold for designing tumor-homing molecules carrying therapeutic or imaging agents (Michnick, S. W., and Sidhu, S. S. (2008) Nat Chem Biol 4(6), 326-329).
Yet, under practical application conditions, an scFv scaffold tends to form aggregation. The aggregation has much to do with the stability of the two variable domains and the dimeric interface. The instability of the scFv structure also compromises the fidelity in reproducing the antibody gene products on phage surface, causing biases in favor of more stable scFv molecules over the less stable ones, or selecting non-folded structures on phage surfaces but nevertheless binding to an antigen. This structural instability thus impacts negatively on the applications of scFv in biotechnology and medical uses.
One way to stabilize the scFv scaffold is to engineer a disulfide bond between the two Fv domains, so that the variable domains can be covalently linked with a disulfide bond. Single chain disulfide-stabilized Fv fragment (sc-dsFv) format was constructed in a single polypeptide chain, as in scFv, with a disulfide framework region (Young, N. M. et al., (1995) FEBS Lett 377(2), 135-139; Worn, A., and Pluckthun, A. (1999) Biochemistry 38(27), 8739-8750). The sc-dsFv molecules could be expressed in E. coli, but not be expressed on phage surface or as soluble form secreted by E. coli in a culture medium, mostly due to severely decreased yield because of the introduction of interface cysteines (Worn, A., and Pluckthun, A. (2001) J Mol Biol 305(5), 989-1010).
Up to now, phage-displayed sc-dsFv libraries and their applications have not been established.

BRIEF SUMMARY OF THE INVENTION

The invention provides a methodology to systematically optimize the signal sequences for phage-displayed protein expression, for which the expression with conventional signal sequences was not viable. The optimized signal sequences and related discovering methodologies led to the establishment of phage display systems with the sc-dsFv format, enabling the demonstration and comparison of the performance of the sc-dsFv phage display platform with that of the conventional scFv platform.
Accordingly, in one aspect, the present invention provides a nucleic acid library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody. The library includes 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 is located 3′ downstream to the first nucleotide. The signal peptide has the amino acid sequence of:
(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:1),
(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:2), or
(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:3).
Each of X₁-X₁₀in (a), (b), and (c) is one of the 20 naturally occurring amino acid residues.
In another aspect, the invention provides a host cell library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody. The library includes 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 is located 3′ downstream to the first nucleotide; the signal peptide has the amino acid sequence of (a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:1),
(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:2), or
(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:3).
each of X₁-X₁₀in (a), (b), and (c) is one of the 20 naturally occurring amino acid residues.
In another aspect, the invention provides a phage library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody. The library has a plurality of phage particles each containing a disulfide-stabilized single chain antibody fused with a coat protein on the surface of the phage. 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; 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; the signal peptide has the amino acid sequence of (a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:1),
(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:2), or
(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:3),
each of X₁-X₁₀in (a), (b), and (c) being one of the 20 naturally occurring amino acid residues.
In addition, the invention provides a sc-dsFv phage display platform. According to the invention, a large scale screening for optimal signal sequences was carried out. In one example of the invention, the signal sequences that were effective for phage-displayed sc-dsFv and non-fusion soluble sc-dsFv secretion in E. coli Amber suppressor strain ER2738 were screened to obtain the sequence preference patterns emerged from the optimum signal sequences.
In still another aspect, the present invention provides an isolated nucleic acid, having 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 signal peptide has the amino acid sequence of
(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:1), in which X₁is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X₂is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X₃is A, F, G, L, M, P, Q, R, S, T, V, or W; X₄is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₅is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₆is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₈is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:2), in which X₁A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X₂is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₃is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₄is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₅is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₆is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X₇is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X₈is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V, X₉is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or
(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:3), in which X₁is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₅is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X₆is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X₈is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.
In a further aspect, the present invention provides a host cell containing the nucleic acid described above.
In a further more aspect, the present invention provides a phage containing a disulfide-stabilized single chain antibody fused with its coat protein on the surface. The phage is prepared by a method having the steps of: providing the above-described host cell, and culturing the host cell in a medium under conditions allowing expression of the phage.
In further another aspect, the present invention provides a method for producing a disulfide-stabilized single chain antibody. The method includes 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 disulfide-stabilized single chain antibody. 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 signal peptide has the amino acid sequence of:
(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:1), in which X₁is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X₂is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X₃is A, F, G, L, M, P, Q, R, S, T, V, or W; X₄is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₅is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₆is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₈is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:2), in which X₁A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X₂is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₃is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₄is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₅is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₆is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X₇is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X₈is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V, X₉is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or
(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:3), in which X₁is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₅is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X₆is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X₈is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.
In addition, the present invention provides a new signal peptide that facilitates production of disulfide-stabilized single chain antibody, and the nucleic acid encoding the signal peptide.
The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

FIG. 1 is a schema showing the signal sequence in pCANTAB5E and the constructs of DNA libraries to diversify the tentative signal sequence responsible for the expression of the phage-displayed pIII fusion proteins.

FIG. 2 is a set of diagrams showing the results of the increased binding to VEGF for phage-displayed sc-dsFv signal sequence variants enriched from the three libraries after selection/amplification cycles, including (A) after each round of selection/amplification cycle, the values of the binding of the rescued phage to immobilized VEGF as measured with ELISA. The ELISA signal strengths are shown in the y-axis, as functions of the selection/amplification cycle; and (B) the numbers of output phage particles titered after each round of selection/amplification cycle for each of the three libraries; the output phage titers, as shown in the y-axis, were plotted against the number of the selection/amplification cycles.

FIG. 3 is a schema showing the DNA construct of the S5 anti-VEGF sc-dsFv as a pIII fusion protein in the pCANTAB5E phagemid.

FIG. 4 is a diagram showing VEGF-binding strengths of the phage-displayed anti-VEGF sc-dsFv's from various signal sequence variants with or without fXa digestion. Eight variants with maximal fXa digestion resistance from a 96-well ELISA plate containing 96 randomly picked variants were selected from each of the VEGF-binding enriched libraries after the 4^thround of selection/amplification cycle. These variants were cultured and the rescued phages were allowed to bind to immobilized VEGF with (grey histogram) and without (black histogram) the fXa treatment, and the VEGF-binding strengths (y-axis) were measured with ELISA. The error bars were derived from three repeats of the ELISA measurement.

FIG. 5 is a diagram showing the binding strengths of phage-displayed anti-HAs scFv/sc-dsFv. One of the scFv phages with specific binding ability to H5, 8a, and the other one with broad-spectrum ability to HAs, 12a, were engineered to disulfide-stabilized scFv (ds-scFv) formats; the sc-dsFv construct was different from the scFv construct in the mutations (L:Gly100Cys & H:Gly44Cys). Avl was negative control of an scFv displayed on the phage; and TAA means the phage does not contain any displayed protein; and various HA subtypes were precoated to ELISA wells to determined binding activity, and the error bars were derived from three repeats of the ELISA measurements.

FIGS. 6A and B are diagrams showing correlations between sc-dsFv folding quality and resistance to fXa digestion. FIG. 6A shows a comparison of the extents (percentages) of the interface disulfide bond formation of the sc-dsFv from the optimum signal sequence variants from L4; both of the axes show the ratio (percent) of the ELISA signal for VEGF-binding after the fXa treatment over the ELISA signal for VEGF-binding before the fXa treatment; the y-axis shows the data from secreted sc-dsFv; the x-axis shows the data from phage-displayed sc-dsFv. FIG. 6B shows a comparison of the extents (percentages) of the interface disulfide bond formation (y-axis) with the folding quality (x-axis) of the sc-dsFv from the optimum signal sequence variants from L4. The sc-dsFv folding quality (x-axis) is represented as the sc-dsFv-VEGF binding ELISA signal divided by western blot signal probed with anti-E tag antibody (E/W, VEGF binding signal divided by secreted sc-dsFv quantity), and then the ratio is normalized with that of anti VEGF scFv (fXa+) (CE/CW, VEGF binding signal divided by secreted scFv quantity), that is, the folding quality is quantified with the ratio: (E/W)/(CE/CW); the error bars in each data point indicate the standard deviations from three repeats of the experiment; the coefficient of determination R2 and the p-value from Spearman's rank correlation coefficient was shown in each panel.

FIGS. 7A and B are diagrams showing stability test of soluble sc-dsFv; including FIG. 7A showing the results of the soluble sc-dsFv incubated at 37° C. as the indicated time shown in the x-axis, and the binding capacities estimated with ELISA against VEGF, shown in y-axis; the ELISA signal was normalized against that of the secreted protein kept at 4° C.; and FIG. 7B showing the fXa resistance percentages of the soluble sc-dsFv plotted against the end binding capacities after 12 days of incubation in 37° C.; the error bars in each data point indicate the standard deviations from three repeats of the experiment, and the coefficient of determination R2 and the p-value from Spearman's rank correlation coefficient are shown in the panel.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
The articles “a” and “an” are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
Amino acids can be expressed by three letters or one letters. Table 1 lists standard amino acid abbreviations.

TABLE 1

Standard amino acid abbreviations

Amino Acid	3-Letter	1-Letter

Alanine	Ala	A
Arginine	Arg	R
Asparagine	Asn	N
Aspartic acid	Asp	D
Cysteine	Cys	C
Glutamic acid	Glu	E
Glutamine	Gln	Q
Glycine	Gly	G
Histidine	His	H
Isoleucine	Ile	I
Leucine	Leu	L
Lysine	Lys	K
Methionine	Met	M
Phenylalanine	Phe	F
Proline	Pro	P
Serine	Ser	S
Threonine	Thr	T
Tryptophan	Trp	W
Tyrosine	Tyr	Y
Valine	Val	V

Very little is known as to why some sc-dsFv constructs could not be expressed on phage surface, and why the disulfide bonds of the newly synthesized preprotein can only be formed in the oxidizing environment of periplasm. The mechanism for the translocation of the nascent unfolded polypeptide chain from the translation site in the cytoplasm across the periplasmic membrane could be a key determinant for the folding. It was unexpectedly found in the invention that for the expression of the displayed protein on the phage surface, alternative sequences in the signal peptide region can modulate the expression level and folding quality of the displayed protein. Accordingly, the invention provides a methodology to systematically optimize the signal sequences for phage-displayed protein expression. Based on the optimized signal sequences and the methodologies of the invention, phage display systems with the sc-dsFv format are established.
According to the present invention, a nucleic acid library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody is provided. The library has 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 is located 3′ downstream to the first nucleotide. The signal peptide has the amino acid sequence of:
(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:1),
(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:2), or
(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:3),
each of X₁-X₁₀in (a), (b), and (c) being one of the 20 naturally occurring amino acid residues.
In one embodiment, each of the expression constructs further includes a third nucleotide encoding a phage coat protein, and the third nucleotide sequence being located 3′ downstream to the second nucleotide.
The term “signal peptide” or “signal sequence” used herein refers to a short (i.e. 3-60 amino acids long) peptide chain that directs the transport of a protein. The signal peptide is known to be responsible for the sec system-dependent translocation of the sc-dsFv-pIII fusion from the translation site in cytoplasm across the periplasmic membrane, a critical process for the integration of the displayed protein on the recombinant phage. Considering the vast signal peptide sequence space needed to be explored, the present invention provides biological combinatorial strategies to diversify the signal peptide sequences with synthetic phage display libraries. The variants in the phage libraries were selected and screened for high expression capabilities, so as to identify the key regions of the signal peptide sequences, including the optimal amino acid sequences, positions and types that are effectively responsible for the sc-dsFv expression on phage surface.
The term “single chain variable fragment” or “scFv” used herein refers to a single polypeptide chain antibody fragment construct encoding a first variable region and a second variable region, with a flexible linkage peptide connecting the two domains. The first and the second variable region can be either a light chain or a heavy chain variable region. The recombinant antibody fragment frequently retains antigen-recognizing capability rivaling that of the parent antibody. One shortcoming of the scFv scaffold is the aggregation tendency of the scFv molecules under physiological and storage conditions. The aggregation mechanism has much to do with the stability of the two variable domains and the dimeric interface. This structural instability has thus impacted negatively on the utilities of scFv, leading to uncertainties to the outcomes of the selected and screened scFv molecules in terms of their potential applications in biomedicine.
The term “disulfide-stabilized single chain antibody variable fragment” or “sc-dsFv” used herein refers to a single polypeptide chain containing two variable regions capable of forming an interface disulfide bond, where each of the two variable regions may be a heavy chain variable region or a light chain variable region. According to the invention, the sc-dsFv-pIII fusion protein can be prepared by using the optimal signal sequences capable of directing the sc-dsFv expression on phage surface.
In an embodiment of the invention, the overlapping segments encompassing the complete signal sequence region governing the protein trafficking of the model anti-VEGF sc-dsFv fusion protein were searched with biological combinatorial methodology for sequence preferences leading to effective expression of the sc-dsFv. The engineering platform established for the disulfide-stabilized antibody variable domain fragment as demonstrated could be used to prepare many of scFv molecules in a more stable structure, which could be carried out under harsh conditions, and have longer shelf-life.
According to one embodiment of the invention, to select signal sequences for effective expression of anti-VEGF sc-dsFv on M13 phage surface, phage display libraries L2, L3 and L4 were constructed to diversify the signal sequence as shown in FIG. 1, where M13pIII-pelB indicated the signal sequence being the wild type signal sequence for pIII in M13 phage genome in connection with pelB peptidase cleavage site. The complexities of the L2, L3 and L4 phage display library were 3.1×10⁹, 3.7×10⁹, and 1.5×10⁹, respectively. These libraries were designed to efficiently diversify the signal peptide sequences on identifying the optimum signal peptides for expression sc-dsFv.
In one example of the invention, the expression construct is a phagemid. Among the expression constructs, the nucleotide sequence of the signal peptide, sc-dsFv and the phage coat protein could be operatively linked in a random order. In one preferred example of the invention, the second nucleotide sequence encoding sc-dsFv is located 3′ downstream to the first nucleotide encoding the signal peptide, and the third nucleotide sequence encoding the phage coat protein is located 3′ downstream to the second nucleotide sequence.
In one embodiment of the invention, a sc-dsFv library, containing more than one billion sc-dsFv variants, is propagated with an E. coli vector of bacterial phage origin following the method as described by McCafferty, J. et al. (Nature 348(6301), 552-554, 1990). The recombinant phages displaying the sc-dsFv variants can be selected or screened for antigen-binding and re-amplified with the host cells, i.e. E. coli.
Furthermore, the present invention provides a host cell library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody. The library includes a plurality of host cells each containing the aforementioned expression constructs.
The present invention also provides a phage library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody. The library includes a plurality of phage particles each containing a disulfide-stabilized single chain antibody fused with a coat protein on the surface of said phage. 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. The expression construct includes (1) a first nucleotide sequence encoding a signal peptide, (2) 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 (3) a third nucleotide sequence encoding a phage envelop protein, the third nucleotide sequence being located 3′ downstream to the second nucleotide sequence. The signal peptide has the amino acid sequence of
(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:1),
(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:2), or
(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:3),
each of X₁-X₁₀in (a), (b), and (c) is one of the 20 naturally occurring amino acid residues.
On the other hand, a sc-dsFv engineering platform is established for preparation of scFv molecules in a more stable structure in the present invention. Accordingly, the present invention provides an isolated nucleic acid that has 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 is located 3′ downstream to the first nucleotide. The signal peptide has the amino acid sequence of
(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:1), in which X₁is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X₂is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X₃is A, F, G, L, M, P, Q, R, S, T, V, or W; X₄is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₅is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₆is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₈is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:2), in which X₁is A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X₂is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₃is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₄is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₅is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₆is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X₇is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X₈is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X₉is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or
(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:3), in which X₁is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₅is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X₆is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X₈is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.
According to the invention, the nucleic acid further includes a third nucleotide encoding a phage coat protein. The third nucleotide sequence is located 3′ downstream to the second nucleotide sequence.
In one example of the invention, anti-VEGF sc-dsFv phage display platform was developed. As shown in FIG. 1, expression constructs for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody were designed. Each of the three DNA libraries (L2, L3, and L4) contained ten consecutive NNK degenerate codons covering overlapping regions around the signal sequence. N stands for A, G, T, or C, 25% each; K stands for G or T, 50% each. The NNK degenerated codon represents 32 possible triplet combinations, encoding all 20 natural amino acids and an amber stop codon (TAG). Each of the phage display libraries was selected for binding against immobilized VEGF. The trends of enrichment of the VEGF-binding phage variants from each of the three libraries, plotted as functions of the number of selection/amplification cycle, are shown in FIG. 2. The enrichment trends were similar among the variants from the three libraries. This result indicates that the signal sequence regions covered by the three signal sequence libraries (see FIG. 1) can all be optimized to increase the expression of the correctly folded anti-VEGF sc-dsFv on phage surface.
In order to further identify binding variants, more than 3000 colonies were randomly selected from each of the libraries L2, L3, and L4 after selection/amplification cycles for enrichment of the binding variants. These phage colonies were individually rescued and spotted on nitrocellulose membranes coated with VEGF (100 μg/30 ml). According to the invention, each of the signal peptides having the amino acid sequences of SEQ ID NO: 5-683 as listed in Tables 2, 3 and 4 was obtained and proved to be capable of facilitating the expression of the sc-dsFv on phage surface. After normalization based on the standard phage solution signals in each of the blocks, the phage-displayed scFv expression efficiency for each of the samples was calculated with the following equation:
$Ratio = \frac{sample (CV)}{sample (C 0)} / \frac{control (CV)}{control (C 0)}$
The value of the sample (CV) is the average normalized signal from VEGF-coated membrane; that of the sample (CO) is the averaged normalized signal from the un-coated and un-blocked membrane. Similarly, those of the control (CV) and control (CO) are the averaged normalized signals for the control phage in the same block where the sample signals are measured on corresponding membrane. The ratio derived from the equation was used to rank the efficiency of the sample phage binding to the immobilized VEGF. All the phage samples with measurable binding strengths with the immobilized VEGF were ranked; the signal sequences of the top fifty ranked phage samples are shown and marked with “*” in Tables 2-4.
Accordingly, new signal peptides that facilitate production of disulfide-stabilized single chain antibody were obtained (see Example 2). In the embodiment of the invention, the signal peptide selected from the group consisting of the peptides having the amino acid sequences set forth in SEQ ID NOS: 5-683 were proved to facilitate production of disulfide-stabilized single chain antibody. On the other hand, a new isolated nucleic acid encoding the above mentioned signal peptide was provided as well.
In a preferred embodiment of the invention, the signal peptide selected from 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, 471-519 and 566 was obtained and proved to facilitate production of disulfide-stabilized single chain antibody. Accordingly, the preferred isolated nucleic acid encoding each signal peptide as mentioned was also provided.
In one example of the present invention, the anti-VEGF sc-dsFv was developed by using the signal peptides as identified and obtained by the method of the present invention. In another example, anti-H5 sc-dsFv against influenza virus was developed (see FIG. 5).
In order to confirm the formation of disulfide bond in the phage-displayed sc-dsFv variants of the present invention, a fXa substrate sequence (-IEGR-) in the linker sequence between the two variable domains was constructed. As shown in FIG. 4, without the fXa treatment, both anti-VEGF scFv(fXa+) and scFv(fXa−) bound to immobilized VEGF. In contrast, with the fXa treatment, only the anti-VEGF scFv(fXa−) bound to immobilized VEGF. The cleavage of the fXa substrate sequence in the phage-displayed anti-VEGF scFv(fXa+) resulted in separation of the variable domains, which in turn abolished the affinity of the phage-displayed scFv against immobilized VEGF. The anti-VEGF scFv(fXa−) was quite insensitive to the treatment of fXa, indicating that no other fXa substrate sequences exist in the displayed protein.
Unexpectedly, it was found in the present invention that each of the signal peptides having the amino acid sequences of SEQ ID NOS: 5-683 as listed in Tables 2-4 enabled the expression and proper folding of the sc-dsFv structure on the phage-displayed platform. In addition, they resulted in secretion of the soluble non-fusion sc-dsFv in culture media.
Accordingly, the present invention also provides a method for producing a disulfide-stabilized single chain antibody. The method includes 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. 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 is located 3′ downstream to the first nucleotide. The signal peptide has the amino acid sequence of:
(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:1), in which X₁is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X₂is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X₃is A, F, G, L, M, P, Q, R, S, T, V, or W; X₄is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₅is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₆is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₈is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:2), in which X₁A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X₂is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₃is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₄is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₅is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₆is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X₇is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X₈is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X₉is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or
(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:3), in which X₁is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₅is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X₆is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X₈is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.
Similar to the aforementioned experiment, the extent (percentage) of the interface disulfide bond formation of the sc-dsFv from the optimum signal sequence variants from L4 were tested. As shown in FIG. 6A, signal sequence optimization could improve the disulfide bond formation in the sc-dsFv from ˜0% up to 40% of the secreted sc-dsFv molecule. As shown in FIG. 6B, the interface disulfide bond formation enhanced the affinity for the sc-dsFv-VEGF interaction.
In the present invention, a stability test of soluble sc-dsFv was conducted. As shown in FIGS. 7A and 7B, the sc-dsFv antibody fragment scaffold was indeed substantially more stable than the scFv scafold due to the interface disulfide bond in the sc-dsFv constructs.
According to the invention, the concentration of sc-dsFv antibody produced by the method disclosed herein was unexpectedly high, and stable. Thus, the present invention provides the sc-dsFv at a high concentration sufficient for coating on a solid phase to produce an array for detection or diagnosis without aggregation, different from the prior art where sc-Fv tends to precipitate under the same concentration due to aggregation.
Accordingly, the present invention provides an array of disulfide-stabilized single chain antibodies produced by the aforementioned method coated on a solid phase. In one example of the invention, the solid phase may be made from silicon, plastic, nylon, glass, ceramic, photoresist or rubber. In one embodiment of the present invention, a microarray test was established using the disulfide-stabilized single chain antibodies produced by the method of the invention, demonstrating that influenza virus could successfully be detected by an array of a serious dilution of anti-H5 sc-dsFv coated on a glass.
The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation.

EXAMPLES

Preparation

1

VEGF Expression and Purification—Human VEGF-121

Human VEGF-121 (VEGF-A residue 34-135 receptor binding domain) (Fuh, G. et al., (2006) J. Biol. Chem., 281, 6625-6631) was expressed in E. coli as inclusion body. The refolding and purification of VEGF-A were carried out as described in Chang, H. J., et al., (2009) Structure, 17, 620-631.

Preparation 2

Phage Display Libraries with Diversified Signal Sequences N-terminal to the sc-dsFv-pIII Fusion Protein

Phage display libraries with diversified sequences in the signal peptide region N-terminal to the sc-dsFv-pIII fusion protein were constructed with pCANTAB5E phagemid (GE-Amersham Biosciences) as shown in FIG. 1. Primers encoded with the sequence diversification shown in FIG. 1 were synthesized by IDT (Integrated DNA Technologies).
For each of the phage display libraries, phagemid templates were constructed with TAA stop codons inserted in the sequence region for diversification (Huang et al., (2010) J. Biol. Chem., in press). The M13pIII-pelB signal sequence for phage-displayed pIII-fusion protein is a combination of the wild-type M13 signal peptide N-terminal to gene III (MKKLLFAIPLVVPFYSHS) (SEQ ID NO: 684) and the pelB signal sequence of Pectobacterium wasabiae (MKYLLPTAAAGLLLLAAQPAMA) (SEQ ID NO: 685). This merged signal sequence (shown in bold font above) was considered containing the tentative n- h- and c-regions of the signal sequence. DNA libraries were constructed to diversify the amino acid sequence in the key regions. Each of the four of DNA libraries (L2, L3, L7) contained ten consecutive NNK (N stands for 25% of G, C, A, and T, and K stands for 50% of G and T; underlined by dashed lines) degenerate codons covering a portion of the tentative signal sequence. Also shown in the Figure are the sequences containing TAA stop codons (underlined regions) used as the templates for the library constructions. The oligonucleotide-directed mutagenesis procedure initially proposed by Kunkel (Kunkel et al., (1987) Methods Enzymol, 154, 367-382) was used for the phagemid library construction. The TAA stop codons in the phagemid templates ensure that the un-mutated phagemid templates after the mutagenesis procedure are incapable of producing pIII fusion protein for phage surface display (Sidhu and Weiss, (2004) Constructing phage display libraries by oligonucleotide-directed mutagenesis. In: Clackson, T., and Lowman, H. B. (eds). Phage Display, 1st Ed., Oxford University Press, New York).
After the oligonucleotide-directed mutagenesis procedure, E. coli strand ER2738 was transformed with the phagemid libraries and the recombinant phage particles were rescued with helper phage M13KO7 (GE-Amersham Biosciences). The phage particles were precipitated with PEG/NaCl, and resuspended in PBS. More details of the phage library preparation can be found in a previous publication (Hsu, H. J. et al., (2008) J Biol Chem 283(18), 12343-12353).
Seven sc-dsFv variants were constructed on the basis of the phagemid encoding the template anti-VEGF scFv(fXa+): S1(L:Gln38Cys & H:Gln39Cys); S2(L:Gly41Cys & H:Gly42Cys); S3(L:Ala43Cys & H:Gln112Cys); S4(L:Phe98Cys & H:Leu45Cys); S5(L:Gln100Cys & H:Gly44Cys); S6(L:Gln38Cys & H:Leu45Cys); S7(L:Ala43Cys & H:Gln112Cys & L:Gln100Cys & H:Gly44Cys). These cysteine pairs were determined by distance constrain for possible disulfide bonds in the model structure (PDB code: 2FJG).

Preparation 3

Biopanning Against VEGF with Phage-Displayed Anti-VEGF sc-dsFv Libraries

Maxisorb Immune Tubes (Nunc) were coated with VEGF (25 μg in 1 ml PBS in each tube) at 4° C. overnight. The tubes were blocked with 4 ml of 5% skim milk in PBST (PBS with 0.05% Tween 20) for one hour at room temperature with gentle shaking and then washed with PBST. In each of the tubes, 10¹¹colony-forming units (cfu) of phage from each of the phage display libraries were mixed with 1 ml of 5% skim milk. The phage particles were allowed to bind to the immobilized VEGF in the tube at room temperature for two hours under gentle shaking. After the binding, the tubes were washed 10 times with PBST and 2 times with PBS. One milliliter of E. coli strand ER2738 in the log phase was added to each of the tubes at room temperature with gentle shaking for 15 minutes. From each tube, the infected E. coli was transferred to 10 ml of a 2YT medium containing 20 μg/ml of ampicillin and was titered with 2YT agar plates containing 100 μg/ml of ampicillin. The infected E. coli was incubated at 37° C. for one hour with vigorous shaking Ampicillin was then added to reach final concentration of 100 μg/ml. The culture was incubated for another hour at 37° C. before transferred to final 100 ml 2YT medium (100 μg/ml of ampicillin) containing 10¹¹cfu M13KO7 helper phage. After two hours of incubation, kanamycin was added to final concentration of 70 μg/ml. The culture was incubated at 37° C. overnight with vigorous shaking. The phage in the supernatant of the culture was harvested by centrifugation. The phage was titered, precipitated with PEG/NaCl, and resuspended in PBS. The phage solution was ready for the next round of selection.

Preparation 4

Enzyme-Linked Immunosorbent Assay (ELISA) for Phage-Displayed Anti-VEGF sc-dsFv Binding Against Immobilized VEGF and Anti-E-Tag Antibody

Single E. coli colonies harboring the selected phagemids were randomly picked using a GENETIX Qpix II colony picker to 96-well deep well culture plates. Each well contained 960 μl 2YT (100 μg/ml of ampicillin and 10 μg/ml of tetracyclin). The culture plates were incubated at 37° C. shaking vigorously for 4 hours before adding 20 μl of M13KO7 helper phage (10″ cfu/ml). The plates were then incubated at 37° C. for one hour with vigorous shaking before adding 20 μl of kanamycin to the final concentration of 50 μg/ml. After overnight incubation at 37° C. with vigorous shaking, the cultures were centrifuged at 3000 g for 10 minutes at 4° C. From each well of the culture plates, 100 μl of the supernatant was mixed with 100 μl of 5% skim milk. Half of the phage mixture was added to a corresponding well of a 96-well Maxisorb microtiter plate precoated with VEGF (1 μg/well) and blocked with 5% skim milk; the other half was added to a corresponding well of another microtiter plate precoated with polyclonal goat anti-E-tag antibody (1 μg/well, Novus Biologicals). After one hour incubation at room temperature, the ELISA plates were washed six times with PBST. The phage particles remained on the plates were measured with HRP-labeled mouse anti-M13 antibody (1/3000, GE Healthcare) and TMB substrate (KPL). The reaction was stopped with 50 μl of 1 N HCl and the signal intensity was measured at OD 450 nm.

Preparation 5

Measurement of Interface Disulfide Bond Formation in Phage-Displayed Anti-VEGF sc-dsFv

Fifty microliters of a freshly prepared phage supernatant (see above) was mixed with 50 μl of a two-fold concentrated reaction buffer containing 1 unit of bovine factor Xa (fXa) (Novagen) in a Maxisorb microtiter plate precoated with VEGF (1 μg/well) and blocked with 5% skim milk. After two hours of enzymatic reaction at 37° C., the phage particles remained bound to the microtiter plate were measured following the same ELISA procedure as described above.

Preparation 6

Western Blot Assay for the Phage-Displayed Anti-VEGF sc-dsFv

Single colony phage was amplified, harvested, precipitated with PEG/NaCl, and resuspended in PBS (see above). Phage particles (10¹¹cfu) were prepared under either a non-reducing or reducing condition before electrophoresis in a 10% SDS-polyacrylamide gel. After the electrophoresis, the proteins in the gel were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was blocked with 5% skim milk for 1 hour at room temperature and then incubated with a monoclonal mouse anti-pIII antibody (1/3000 mg/ml, New England Biolabs) for one hour at room temperature. After three washes (5 minutes each) with PBST, the membrane was incubated with HRP-labeled anti-mouse antibody (1/3000, GE Healthcare) for 1 hour at room temperature. After three washes with 10 ml PBST, the membrane was developed with 4-chloro-1-naphthol (4CN) substrate (KPL) until the desired color intensity was achieved.

Preparation 7

Preparation of Non-Fusion Soluble scFv/sc-dsFv

Seven hundred and fifty microliters of mid-log phase (OD_600nm=0.6) E. coli host (non-suppressor strain HB2151 or suppressor strain ER2738) grown in a 2YT medium (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, pH 7.0) was infected with 50 μl of a phage solution (10¹¹cfu/ml). After one hour incubation at 37° C. with shaking, 100 μl ampicillin in a 2YT medium was added to the final concentration of 100 μg/ml. After another hour of incubation, 100 μl isopropyl-beta-D-thiogalactopyranoside (IPTG) in a 2YT medium was added to the final concentration of 1 mM. The culture was kept at 37° C. with vigorous shaking overnight. The secreted soluble scFv or sc-dsFv in the supernatant was separated from the bacterial host by centrifugation at 3000 g for 10 minutes.

Preparation 8

ELISA for Immobilized VEGF Binding

For phage ELISA, each well in a Maxisorb 96-well microtiter plate (Nunc) was coated with 2 μg VEGF at 4° C. overnight. The wells were blocked with 5% skim milk in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, 0.1% tween20, pH 7.4) for one hour. After 3×300 μl PBST and 2×300 μl PBS washes, 100 μl of a phage solution and 100 μl of 5% skim milk in PBST were added to each well and incubated at room temperature with shaking for one hour. After washing each of the wells three times with 300 μl of PBST each and twice with 300 μl of PBS each, the bound phages were labeled with anti-M13 antibody conjugated with HRP (GE-Amersham) 1/3000 dilution in 5% skim milk in PBST for one hour. The ELISA signal was developed by incubating each well with 100 μl of a TMB solution (KPL Inc.) for 5 minutes. The reaction was stopped with 100 μl 1 N HCl, and the optical density was recorded with VICTOR3 Multilabel Plate Readers (Perkin Elmer) at 450 nm.
For scFv or sc-dsFv ELISA, 100 μl of a soluble scFv solution was used instead of phage solution, and HRP-conjugated protein L (0.5 μg/ml in 5% skim milk in PBS, from Pierce) was used instead of HRP-conjugated anti-M13 antibody. When needed, the ELISA signals were normalized with the signals of the control anti-VEGF scFv in serial dilution.

Preparation 9

fXa Protease Digestion

For phage solutions, 20 μl (1 unit) of bovine factor Xa (fXa) protease (Novagen) in a six-fold concentrated reaction buffer was added to 100 μl of a phage solution at 37° C. After 2 hours of enzymatic reaction, 100 μl of 5% skim milk in PBST was added to the reaction mixture before the VEGF-binding ELISA measurement was carried out in the manner described in the previous section. The fXa resistance percentage was calculated with the ratio of the ELISA reading in the presence of fXa over the ELISA reading in the absence of fXa. The ELISA readings for the ratio were adjusted by shift the baseline determined with the null control ELISA readings. For soluble scFv/sc-dsFv fXa digestion, all procedures were the same except that the enzymatic reaction was carried out for one hour at room temperature.

Preparation 10

Construction of anti-H5 sc-dsFv against Influenza Virus

The construction of scFv library derived from mouse spleen after immunization of hemagglutinin from influenza virus was based on the protocols described in “Phage Display, A Laboratory Manual, edited by Carlos F. Barbas Ill., Dennis R. Burton, Jamie K. Scott, and Gregg J. Silverman, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA”. In brief, after hemagglutinin immunization, the total RNA derived from mouse spleen was purified by Trizol reagent (Invitrogen) according to the manufacturer's protocols. After cDNA synthesis by reverse transcriptase, the gene fragments encoded heavy and light chains of antibody variable region were amplified by the specific primer sets described in the book mentioned above, respectively. The scFv fragments were synthesized by two-steps PCR reactions, and then cloned into a pCANTAB 5E phagemid vector with the signal sequence derived from the library 2 for sc-dsFv phage production. The library complexity was 4.5×10⁷. After panning against H5, two clones were selected for mono-spectral binding to H5 (clone 8a) and broad spectral binding to H1, H3 and H5 (clone 12a). These two clones were subjected to disulfide bond formation mutants between L100 and H44 (based on Kabat numbering) and then for sc-dsFv phage production and ELISA detection (8aS5 and 12aS5, respectively).

Preparation 11

Microarray Test for sc-dsFv Binding of H5 Influenza Virus

The just-described sc-dsFv ds-8a or and ds-12a was subcloned into a pET32a vector with thioredoxin as a fusion protein partner at the N-terminus. The thioredoxin-sc-dsFv fusion proteins could be expressed in Rosetta-gami B strain of E. coli in a soluble form. After purification and TEV protease digestion to remove thioredoxin, the purified ds-8a protein was found to have a binding affinity and specificity similar to those of H5 based on an ELISA assay and array studies. The protein ds-AV1, ds-12a and ds-8a were spot on glass slides coated with streptavidin as the purified ds-AV1, ds-12a and ds-8a proteins contained a biotinylated Avitag sequence at their C-termini. The highest protein concentration used for this protein array was 8 mg/ml, 8 mg/ml, and 0.8 mg/ml, respectively. These proteins were 2 fold dilution with a 100 mM sodium phosphate buffer, pH 8.5 from the highest protein concentration for 15 times for spotting (10 nl/spot), and then the protein of each concentration was spotted for 5 replicates. After spotting, each glass slide was sealed to form 16 distinct squares for reaction. After blocking with 5% BSA for 30 minutes, the H5 influenza virus (about ˜10⁷PFU/ml) was added to react with spotted sc-dsFvs for 30 minutes. After 3 times wash with a phosphate buffer for 5 minutes each, 40 nm fluorescence beads coated with ds-8a (˜10⁷) were added to each square and incubated for 30 minutes. After 3 times wash again with a phosphate buffer for 5 minutes each, the glass array was air-dried for detection.

Example 1

Selection of Signal Sequences for Effective Expression of anti-VEGF sc-dsFv on M13 Phage Surface

Phage display libraries L2, L3, and L4 were constructed to diversify the signal sequence of the S5 anti-VEGF sc-dsFv-pIII fusion protein as shown in FIG. 1. The complete DNA construct and the amino acid sequence of the S5 anti-VEGF sc-dsFv are shown in FIG. 1. The S5 sequence remained unchanged in all the variants from the libraries. The complexities of the L2, L3, and L4 phage display libraries were 3.1×10⁹, 3.7×10⁹, and 1.5×10⁹, respectively. These libraries were designed to diversify the signal peptide sequences in the h-region, c-region, and a few N-terminal residues of the mature phage-displayed anti-VEGF sc-dsFv.
Each of the phage display libraries was selected for binding against immobilized VEGF. The trends of enrichment of the VEGF-binding phage variants from each of the three libraries, plotted as functions of the number of selection/amplification cycle, are shown in FIG. 2. After four rounds of selection/amplification cycle, the VEGF-binding phage variants were enriched for more than one order of magnitude. The enrichment trends were similar among the variants from the three libraries. This result indicates that the signal sequence regions covered by the three signal sequence libraries (FIG. 1) could all be optimized to increase the expression of the correctly folded anti-VEGF sc-dsFv on phage surface.

Example 2

Interface Disulfide Bond Formation in anti-VEGF sc-dsFv on M13 Phage Surface

In order to test the formation of the disulfide bond in the phage-displayed sc-dsFv variants, we constructed two control phage-displayed anti-VEGF scFv variants: one with a factor Xa cutting site, -IEGR-, encoded in the linker peptide connecting the two variable domains (anti-VEGF scFv(fXa+)); the other without this fXa cutting site (anti-VEGF scFv(fXa−)). As shown in FIG. 3, the S5 anti-VEGF sc-dsFv was constructed with a fXa substrate sequence (-IEGR-) in the linker sequence between the two variable domains. The cleavage of the fXa substrate sequence in the phage-displayed anti-VEGF scFv(fXa+) resulted in separation of the variable domains, which in turn abolished the affinity of the phage-displayed scFv against immobilized VEGF. Both phage-displayed scFv's did not have the engineered interface disulfide bond as in S5; the scFv(fXa+) construct had the -IEGR- site in the linker peptide (-(G)₄SIEGRS(G)₄S—), while the scFv(fXa−) construct had the conventional -(G)₄S(G)₄S(G)₄S— linker peptide.
As shown in FIG. 4, without the fXa treatment, both anti-VEGF scFv(fXa+) and scFv(fXa−) bound to immobilized VEGF. But with the fXa treatment, only the anti-VEGF scFv(fXa−) bound to immobilized VEGF. In contrast, all the S5 signal sequence variants for the phage-displayed sc-dsFv showed substantial increase in resistance to fXa protease activity, indicating that the interface disulfide bonds in the anti-VEGF sc-dsFv's were formed to stabilize the functional dimeric structure after the cleavage of the peptide linker between the two variable domains. The results unambiguously demonstrated that the engineered interface disulfide bond was correctly formed in the phage-displayed S5 anti-VEGF sc-dsFv from some of the signal sequence variants from all three VEGF-binding enriched signal sequence libraries (L2, L3 and L4).

Example 3

Preference Sequence Patterns of the Optimum Signal Peptides in Effective Expression of Functional Anti-VEGF sc-dsFv

The functionality of the anti-VEGF sc-dsFv on phage surface was quantified with two quantitative measurements: the affinity of the sc-dsFv against VEGF and the extent of the interface disulfide bond formation. After the tests, 250, 126, and 213 optimum signal sequences were found in L2, L3, and L4 library, respectively, which are summarized as following Tables 2-4. Among them, fifty signal sequence variants with the highest sc-dsFv-VEGF binding affinities selected from more than 3000 random single colonies of the enriched libraries L2, L3, and L4 were marked with the symbol “*.” The symbol “q” indicated that the nucleotide sequence TAG (amber stop codon) that could be translated to Gln (Q) with 0.8˜20% in E. coli amber suppressor strains which were normally used in phage production.

TABLE 2

Preference sequence patterns selected from
L2 S5 sc-dsFv library

			SEQ
			ID
No.	Code	Sequence	NO:

	M13-pe1B	VKKLL	FAIPLVVPFY	AAQPAMAHHHHHH	4

1	1.12B	VKKLL	VLSHLPFMTD	AAQPAMAHHHHHH *	5

2	9.26.10B	VKKLL	SHWLLSSqLQ	AAQPAMAHHHHHH *	6

3	2.12A	VKKLL	AMSLAPSVFP	AAQPAMAHHHHHH *	7

4	9.12A	VKKLL	WSLFFqqLNP	AAQPAMAHHHHHH *	8

5	2.12F	VKKLL	LLSLLQRPLP	AAQPAMAHHHHHH *	9

6	1.2H	VKKLL	LSSWLMTRFP	AAQPAMAHHHHHH *	10

7	6.9G	VKKLL	VLSHFPAFVP	AAQPAMAHHHHHH *	11

8	1.8F	VKKLL	PLLSLPLPPN	AAQPAMAHHHHHH *	12

9	7.1B	VKKLL	VLTPMHFSSP	AAQPAMAHHHHHH *	13

10	9.26.10A	VKKLL	ILALPQSYPL	AAQPAMAHHHHHH *	14

11	5.4A	VKKLL	qALYFSLPSS	AAQPAMAHHHHHH *	15

12	YJ2.2	VKKLL	VSAMTSASFP	AAQPAMAHHHHHH *	16

13	5.2F	VKKLL	LPASWLFGQP	AAQPAMAHHHHHH	17

14	10.2D	VKKLL	WSLFFqqLNP	AAQPAMAHHHHHH *	18

15	YJ2.34	VKKLL	FVMALRSSAP	AAQPAMAHHHHHH *	19

16	3.3F	VKKLL	FLWPFYNGHI	AAQPAMAHHHHHH	20

17	4.1A	VKKLL	QSFYLSLqLD	AAQPAMAHHHHHH *	21

18	10.7H	VKKLL	SLTFPFTIHS	AAQPAMAHHHHHH *	22

19	1.9D	VKKLL	WPVLSPSLFP	AAQPAMAHHHHHH *	23

20	5.12D	VKKLL	PWLFSTFPSS	AAQPAMAHHHHHH *	24

21	1.8D	VKKLL	IMSSLPTLSP	AAQPAMAHHHHHH *	25

22	4.11F	VKKLL	IMSRVLAPDF	AAQPAMAHHHHHH *	26

23	1.7C	VKKLL	FDFWFSSFLq	AAQPAMAHHHHHH *	27

24	4.8G	VKKLL	YGqLMLLSSD	AAQPAMAHHHHHH *	28

25	4.4E	VKKLL	PWLFPFHAYP	AAQPAMAHHHHHH *	29

26	1.12G	VKKLL	LVMTLSRQPF	AAQPAMAHHHHHH	30

27	4.8A	VKKLL	ASAYLYHGLS	AAQPAMAHHHHHH *	31

28	4.4C	VKKLL	PFFAGVLqHP	AAQPAMAHHHHHH *	32

29	3.11A	VKKLL	ALSSPFFHIP	AAQPAMAHHHHHH *	33

30	10.3F	VKKLL	PTRqPMMYPP	AAQPAMAHHHHHH *	34

31	YJ2.15	VKKLL	QLLMPFLNSP	AAQPAMAHHHHHH *	35

32	9.9H	VKKLL	CSLGYACIPP	AAQPAMAHHHHHH *	36

33	4.9C	VKKLL	LMPWLFNSPP	AAQPAMAHHHHHH	37

34	3.12B	VKKLL	LDqLAYAALS	AAQPAMAHHHHHH *	38

35	4.10G	VKKLL	qSTVFFSWLS	AAQPAMAHHHHHH *	39

36	YJ2.18	VKKLL	LPWALSHQVL	AAQPAMAHHHHHH *	40

37	7.2E-q	VKKLL	ALTYPAFLYD	AAQPAMAHHHHHH *	41

38	1.11A	VKKLL	AMAPPMMSMN	AAQPAMAHHHHHH *	42

39	5.3D	VKKLL	WWSSLFAPSP	AAQPAMAHHHHHH	43

40	4.6H	VKKLL	GSFILARSMD	AAQPAMAHHHHHH	44

41	5.11C	VKKLL	MVLTSWHPYP	AAQPAMAHHHHHH *	45

42	2.8C	VKKLL	FSLRFFFPSS	AAQPAMAHHHHHH	46

43	2.5F	VKKLL	WLWSTPLFPH	AAQPAMAHHHHHH	47

44	2.2A	VKKLL	PLLFSLDGDP	AAQPAMAHHHHHH *	48

45	3.2C-d	VKKLL	SVSLSSYSFY	AAQPAMAHHHHHH *	49

46	3.1H	VKKLL	LNGTFSAqLF	AAQPAMAHHHHHH *	50

47	6.4A	VKKLL	WHVLPYLPNS	AAQPAMAHHHHHH *	51

48	4.10E	VKKLL	SIVPLFSPqS	AAQPAMAHHHHHH *	52

49	7.4H	VKKLL	VMTSPMLAPG	AAQPAMAHHHHHH *	53

50	2.5H	VKKLL	VLSLPSIAPH	AAQPAMAHHHHHH	54

51	6.4E	VKKLL	qSLLLLRALL	AAQPAMAHHHHHH *	55

52	2.1A	VKKLL	FSLPVFFDLP	AAQPAMAHHHHHH	56

53	4.11D	VKKLL	LLFSMARPLP	AAQPAMAHHHHHH *	57

54	7.10A	VKKLL	TqAVFPFTFN	AAQPAMAHHHHHH *	58

55	3.2E	VKKLL	LASWLFRADM	AAQPAMAHHHHHH *	59

56	5.2E	VKKLL	PFLFPFPSPS	AAQPAMAHHHHHH *	60

57	YJ2.128	VKKLL	ALSAWSLSQT	AAQPAMAHHHHHH *	61

58	4.7H	VKKLL	ALLPLFPTqH	AAQPAMAHHHHHH *	62

59	2.10F	VKKLL	AALASFPPAP	AAQPAMAHHHHHH *	63

60	YJ2.22	VKKLL	LLMPFLNQSP	AAQPAMAHHHHHH *	64

61	7.5A	VKKLL	FTSGLKLVPP	AAQPAMAHHHHHH	65

62	6.10F	VKKLL	LqPLLSIYLN	AAQPAMAHHHHHH	66

63	4.11B	VKKLL	LSSLWSAYMD	AAQPAMAHHHHHH	67

64	2.5C	VKKLL	LLGqSLMHFQ	AAQPAMAHHHHHH	68

65	YJ2.25	VKKLL	PQLAMSLPSI	AAQPAMAHHHHHH	69

66	10.3H	VKKLL	YETMLSSYLY	AAQPAMAHHHHHH	70

67	3.10D	VKKLL	SLYYFPLVPY	AAQPAMAHHHHHH	71

68	4.7C	VKKLL	qRTVAAAYFW	AAQPAMAHHHHHH	72

69	4.12D	VKKLL	FLTWLRYGFP	AAQPAMAHHHHHH	73

70	6.1A	VKKLL	LLLTLMqPTS	AAQPAMAHHHHHH	74

71	8.10C	VKKLL	FDFFTHVHLF	AAQPAMAHHHHHH	75

72	5.6E	VKKLL	ALYPHFVSFT	AAQPAMAHHHHHH	76

73	4.11E	VKKLL	LPYAIqLFSP	AAQPAMAHHHHHH	77

74	YJ2.5	VKKLL	WFPLHSSLLP	AAQPAMAHHHHHH	78

75	4.7A	VKKLL	PALLLATAAF	AAQPAMAHHHHHH	79

76	3.11C	VKKLL	LASVAWNLDS	AAQPAMAHHHHHH	80

77	YJ2.121	VKKLL	VGSLLFWPQQ	AAQPAMAHHHHHH	81

78	4.5F	VKKLL	SPLLFLqNYT	AAQPAMAHHHHHH	82

79	3.2F	VKKLL	SYWLDFIqVL	AAQPAMAHHHHHH	83

80	10.3C	VKKLL	VPSFLLSPSP	AAQPAMAHHHHHH	84

81	9.23.7H	VKKLL	SLYWLTSqPL	AAQPAMAHHHHHH	85

82	3.9A	VKKLL	FALSSVHSPP	AAQPAMAHHHHHH	86

83	4.11H	VKKLL	SYYSLLYSYP	AAQPAMAHHHHHH	87

84	3.1C	VKKLL	LVSGLqPWYF	AAQPAMAHHHHHH	88

85	2.5A	VKKLL	VLATPLHLSP	AAQPAMAHHHHHH	89

86	10.6H-q	VKKLL	SLAFPLFTPP	AAQPAMAHHHHHH	90

87	3.6A	VKKLL	SLVPIFPFST	AAQPAMAHHHHHH	91

88	8.10D	VKKLL	qPVLFSFFIR	AAQPAMAHHHHHH	92

89	4.3B	VKKLL	MSqFLNLLSP	AAQPAMAHHHHHH	93

90	2.3G	VKKLL	WAVqPLFPLN	AAQPAMAHHHHHH	94

91	5.3H	VKKLL	MFSLVPSPPI	AAQPAMAHHHHHH	95

92	10.7B	VKKLL	PFFLQPFqFP	AAQPAMAHHHHHH	96

93	7.2D-q	VKKLL	PDLLASVLPV	AAQPAMAHHHHHH	97

94	2.9H	VKKLL	FWqFLWPSLP	AAQPAMAHHHHHH	98

95	6.4A	VKKLL	LLGqFFPNPM	AAQPAMAHHHHHH	99

96	6.4D	VKKLL	TLSALSQWHP	AAQPAMAHHHHHH	100

97	9.4D	VKKLL	SLVYFFPFYP	AAQPAMAHHHHHH	101

98	10.2H	VKKLL	FAFAPAPFYH	AAQPAMAHHHHHH	102

99	4.12B	VKKLL	FLPFALVPRQ	AAQPAMAHHHHHH	103

100	4.1F	VKKLL	ALWMqLYPQD	AAQPAMAHHHHHH	104

101	YJ2.27	VKKLL	ASILFSHAAP	AAQPAMAHHHHHH	105

102	2.2C	VKKLL	LPLPWSLHLY	AAQPAMAHHHHHH	106

103	4.9C	VKKLL	LPHFMSFWFE	AAQPAMAHHHHHH	107

104	7.3E	VKKLL	LFQPFWPIPY	AAQPAMAHHHHHH	108

105	4.7F	VKKLL	LLFSLGRLPP	AAQPAMAHHHHHH	109

106	7.12G	VKKLL	PLWVLLKDPL	AAQPAMAHHHHHH	110

107	9.3B	VKKLL	MSFATLFPHN	AAQPAMAHHHHHH	111

108	4.5B	VKKLL	qHSLVTSWLC	AAQPAMAHHHHHH	112

109	5.2H	VKKLL	LLFqGAFVGq	AAQPAMAHHHHHH	113

110	4.4C	VKKLL	WMFHSLPFSP	AAQPAMAHHHHHH	114

111	6.8G	VKKLL	LTqLLLTRLH	AAQPAMAHHHHHH	115

112	4.10A	VKKLL	ALTLVPSSYP	AAQPAMAHHHHHH	116

113	4.5D	VKKLL	LPWYMLLSDS	AAQPAMAHHHHHH	117

114	9.3E	VKKLL	VVTqFWPSLP	AAQPAMAHHHHHH	118

115	4.3G	VKKLL	LSTLFLWHVR	AAQPAMAHHHHHH	119

116	9.7E	VKKLL	RSLFFqqLYP	AAQPAMAHHHHHH	120

117	YJ2.30	VKKLL	TLTTLHQTFP	AAQPAMAHHHHHH	121

118	1.3B	VKKLL	SALLAPWYWD	AAQPAMAHHHHHH	122

119	8.9B	VKKLL	AIqqRMQIYT	AAQPAMAHHHHHH	123

120	3.4E	VKKLL	LLFPWFQPPY	AAQPAMAHHHHHH	124

121	9.23.7E	VKKLL	YFTSLLGqFP	AAQPAMAHHHHHH	125

122	6.3D	VKKLL	PVLIFLSEIR	AAQPAMAHHHHHH	126

123	9.5G	VKKLL	VATSLRWAVT	AAQPAMAHHHHHH	127

124	YJ2.54	VKKLL	AQLFHLFATH	AAQPAMAHHHHHH	128

125	8.6G	VKKLL	LqFSALFNSF	AAQPAMAHHHHHH	129

126	7.12C-q	VKKLL	FHLMSMLPPP	AAQPAMAHHHHHH	130

127	5.4C	VKKLL	PVCSqSMFPI	AAQPAMAHHHHHH	131

128	YJ2.48	VKKLL	LLLSSSYQSP	AAQPAMAHHHHHH	132

129	4.3D	VKKLL	LDSLFFHAPL	AAQPAMAHHHHHH	133

130	7.7A	VKKLL	qAWVFSAHQL	AAQPAMAHHHHHH	134

131	YJ2.99	VKKLL	FQALGALTSP	AAQPAMAHHHHHH	135

132	9.9D	VKKLL	CFFFFLqFHP	AAQPAMAHHHHHH	136

133	4.12F-f	VKKLL	CFSHLALPSP	AAQPAMAHHHHHH	137

134	6.2B	VKKLL	FGSWIPFTQM	AAQPAMAHHHHHH	138

135	4.6F	VKKLL	GLGYFNWTLL	AAQPAMAHHHHHH	139

136	10.4A	VKKLL	HLFPLFQFHH	AAQPAMAHHHHHH	140

137	5.6B	VKKLL	SEHVSSICVL	AAQPAMAHHHHHH	141

138	3.11E	VKKLL	FSCLLDPTCP	AAQPAMAHHHHHH	142

139	8.3F	VKKLL	LYLLHPSFLP	AAQPAMAHHHHHH	143

140	2.2F	VKKLL	WCAPLLYSLR	AAQPAMAHHHHHH	144

141	2.3F	VKKLL	FAMFPYTFqT	AAQPAMAHHHHHH	145

142	10.5D	VKKLL	LPSLFYVESL	AAQPAMAHHHHHH	146

143	8.8B	VKKLL	SLWLSSLSVL	AAQPAMAHHHHHH	147

144	YJ2.17	VKKLL	PHLWFLWSLK	AAQPAMAHHHHHH	148

145	7.5B	VKKLL	ASDPVWYFLW	AAQPAMAHHHHHH	149

146	10.12D	VKKLL	GLPLMGLqSL	AAQPAMAHHHHHH	150

147	2.4H	VKKLL	PQLLLLRALS	AAQPAMAHHHHHH	151

148	5.5D	VKKLL	APSAFSLHLF	AAQPAMAHHHHHH	152

149	9.4C	VKKLL	FqLSSLFVPY	AAQPAMAHHHHHH	153

150	4.5H	VKKLL	VPSFLSTMIE	AAQPAMAHHHHHH	154

151	2.7B	VKKLL	ASPFFASYLW	AAQPAMAHHHHHH	155

152	YJ2.23	VKKLL	LQYLLSPIGY	AAQPAMAHHHHHH	156

153	6.2D	VKKLL	VLSVPISAHH	AAQPAMAHHHHHH	157

154	7.4A	VKKLL	MMqALSSLPE	AAQPAMAHHHHHH	158

155	4.12B	VKKLL	MPAVLATRLT	AAQPAMAHHHHHH	159

156	6.12E	VKKLL	PFTAWIIDGW	AAQPAMAHHHHHH	160

157	YJ2.125	VKKLL	TQLLPLWQPL	AAQPAMAHHHHHH	161

158	YJ2.21	VKKLL	LVPSLLPLTQ	AAQPAMAHHHHHH	162

159	10.12B	VKKLL	PIqSCMVIPS	AAQPAMAHHHHHH	163

160	YJ2.35	VKKLL	WSLHLATRLL	AAQPAMAHHHHHH	164

161	6.11H	VKKLL	qQVLLCSTLR	AAQPAMAHHHHHH	165

162	7.3B	VKKLL	LLRYFLDPMY	AAQPAMAHHHHHH	166

163	10.12A	VKKLL	IPQFLRSHHR	AAQPAMAHHHHHH	167

164	YJ2.6	VKKLL	GVLHLALSLR	AAQPAMAHHHHHH	168

165	4.12C	VKKLL	LVTSqFSLVP	AAQPAMAHHHHHH	169

166	YJ2.19	VKKLL	PLALSWFQLR	AAQPAMAHHHHHH	170

167	YJ2.88	VKKLL	QHQWYPTVLM	AAQPAMAHHHHHH	171

168	YJ2.29	VKKLL	LMYWLSKPLS	AAQPAMAHHHHHH	172

169	YJ2.8	VKKLL	TQLTLSSSPI	AAQPAMAHHHHHH	173

170	YJ2.94	VKKLL	QLTALLSRLI	AAQPAMAHHHHHH	174

171	YJ2.107	VKKLL	LMTFGTTPQS	AAQPAMAHHHHHH	175

172	YJ2.133	VKKLL	SAFSFSLSST	AAQPAMAHHHHHH	176

173	6.1A	VKKLL	APWLVLPHFP	AAQPAMAHHHHHH	177

174	YJ2.81	VKKLL	HVLSFAPPMP	AAQPAMAHHHHHH	178

175	YJ2.38	VKKLL	NWLFFAHPFS	AAQPAMAHHHHHH	179

176	YJ2.20	VKKLL	QLAVLLGSLR	AAQPAMAHHHHHH	180

177	7.1D	VKKLL	LFGLFYFRAC	AAQPAMAHHHHHH	181

178	YJ2.98	VKKLL	FQFFVVWRLL	AAQPAMAHHHHHH	182

179	YJ2.39	VKKLL	PWAWPPPPFW	AAQPAMAHHHHHH	183

180	YJ2.130	VKKLL	LQLVIVYYLR	AAQPAMAHHHHHH	184

181	YJ2.16	VKKLL	RQSVLLSALH	AAQPAMAHHHHHH	185

182	3.12E	VKKLL	VYGYFLTTFR	AAQPAMAHHHHHH	186

183	YJ2.53	VKKLL	CFSPLFGFHT	AAQPAMAHHHHHH	187

184	YJ2.100	VKKLL	PGYALWQTIP	AAQPAMAHHHHHH	188

185	YJ2.58	VKKLL	QRIFICFFLR	AAQPAMAHHHHHH	189

186	8.2A	VKKLL	PHVFSCqLSA	AAQPAMAHHHHHH	190

187	5.10A	VKKLL	SPLSLSVKLL	AAQPAMAHHHHHH	191

188	9.2D	VKKLL	ARSLFSGSML	AAQPAMAHHHHHH	192

189	YJ2.92	VKKLL	LQFLIVFPLR	AAQPAMAHHHHHH	193

190	YJ2.32	VKKLL	LAVLLGQSLR	AAQPAMAHHHHHH	194

191	YJ2.14	VKKLL	LLSHLFLRLH	AAQPAMAHHHHHH	195

192	8.4E	VKKLL	LAMVFFVTLR	AAQPAMAHHHHHH	196

193	YJ2.117	VKKLL	WLFALPQENV	AAQPAMAHHHHHH	197

194	YJ2.66	VKKLL	HPLVLLSSSP	AAQPAMAHHHHHH	198

195	YJ2.131	VKKLL	LQYLFMLSMR	AAQPAMAHHHHHH	199

196	4.11H	VKKLL	PALLIRYASV	AAQPAMAHHHHHH	200

197	YJ2.78	VKKLL	QQFTSPFLLL	AAQPAMAHHHHHH	201

198	YJ2.44	VKKLL	SPCFFLLYLR	AAQPAMAHHHHHH	202

199	YJ2.90	VKKLL	PGMPLFFTNS	AAQPAMAHHHHHH	203

200	YJ2.47	VKKLL	PQVFFLFRPF	AAQPAMAHHHHHH	204

201	YJ2.110	VKKLL	PFPILLQSPF	AAQPAMAHHHHHH	205

202	YJ2.74	VKKLL	FQACCLFPLQ	AAQPAMAHHHHHH	206

203	YJ2.55	VKKLL	AVVHTMPLFS	AAQPAMAHHHHHH	207

204	YJ2.108	VKKLL	QFSWAFVSIL	AAQPAMAHHHHHH	208

205	YJ2.96	VKKLL	PVCLFWSFFR	AAQPAMAHHHHHH	209

206	YJ2.70	VKKLL	QLLWQQQVPV	AAQPAMAHHHHHH	210

207	YJ2.60	VKKLL	PLQALSWFLR	AAQPAMAHHHHHH	211

208	YJ2.119	VKKLL	FYLLCRLSLQ	AAQPAMAHHHHHH	212

209	YJ2.82	VKKLL	YLQILVICLR	AAQPAMAHHHHHH	213

210	YJ2.63	VKKLL	QLFLIVFPLR	AAQPAMAHHHHHH	214

211	10.5A	VKKLL	PLHFALFFRL	AAQPAMAHHHHHH	215

212	YJ2.85	VKKLL	PFPMHLVLPF	AAQPAMAHHHHHH	216

213	YJ2.86	VKKLL	PLLFSPPSLH	AAQPAMAHHHHHH	217

214	YJ2.126	VKKLL	CQSITFSSIW	AAQPAMAHHHHHH	218

215	YJ2.112	VKKLL	WQRLFPFLLI	AAQPAMAHHHHHH	219

216	YJ2.77	VKKLL	MVPFWPFSFT	AAQPAMAHHHHHH	220

217	YJ2.103	VKKLL	QAFPLPPLLV	AAQPAMAHHHHHH	221

218	YJ2.134	VKKLL	PLYLLFRSFV	AAQPAMAHHHHHH	222

219	YJ2.91	VKKLL	HRSMYLSWLY	AAQPAMAHHHHHH	223

220	YJ2.64	VKKLL	LLSTLVRAPY	AAQPAMAHHHHHH	224

221	YJ2.87	VKKLL	PLALSQWFLR	AAQPAMAHHHHHH	225

222	YJ2.116	VKKLL	AQGMIFFLRL	AAQPAMAHHHHHH	226

223	YJ2.62	VKKLL	FCCRLALQFF	AAQPAMAHHHHHH	227

224	YJ2.102	VKKLL	YLQFLSLMLS	AAQPAMAHHHHHH	228

225	YJ2.106	VKKLL	CQATFPTLLC	AAQPAMAHHHHHH	229

226	YJ2.124	VKKLL	ARSYLYFSLS	AAQPAMAHHHHHH	230

227	YJ2.111	VKKLL	YQSSFLPLFW	AAQPAMAHHHHHH	231

228	YJ2.104	VKKLL	SASFLAFRIT	AAQPAMAHHHHHH	232

229	YJ2.67	VKKLL	SVLFLSHYHS	AAQPAMAHHHHHH	233

230	YJ2.105	VKKLL	PLALLYVRLS	AAQPAMAHHHHHH	234

231	YJ2.127	VKKLL	PEFLLLFRFF	AAQPAMAHHHHHH	235

232	YJ2.80	VKKLL	FPSLYAWGGL	AAQPAMAHHHHHH	236

233	YJ2.122	VKKLL	LQAAAFFCWL	AAQPAMAHHHHHH	237

234	YJ2.79	VKKLL	PFFLFCSSLR	AAQPAMAHHHHHH	238

235	YJ2.115	VKKLL	ELTQLWLFHL	AAQPAMAHHHHHH	239

236	YJ2.113	VKKLL	PGVPLLLCFR	AAQPAMAHHHHHH	240

237	YJ2.114	VKKLL	SQAYLSYFLY	AAQPAMAHHHHHH	241

238	YJ2.61	VKKLL	ISYAFLVRVT	AAQPAMAHHHHHH	242

239	YJ2.123	VKKLL	APALLRSILA	AAQPAMAHHHHHH	243

240	YJ2.109	VKKLL	HSHTLLMSLH	AAQPAMAHHHHHH	244

241	YJ2.83	VKKLL	AVSAFVSLVR	AAQPAMAHHHHHH	245

242	YJ2.31	VKKLL	TLITFKFLPH	AAQPAMAHHHHHH	246

243	YJ2.49	VKKLL	QQFAIPLVEF	AAQPAMAHHHHHH	247

244	YJ2.75	VKKLL	MPCLLVYYLE	AAQPAMAHHHHHH	248

245	YJ2.71	VKKLL	RYCLLLQIVR	AAQPAMAHHHHHH	249

246	YJ2.45	VKKLL	SLALLRVSLG	AAQPAMAHHHHHH	250

247	YJ2.68	VKKLL	IIGRIALILR	AAQPAMAHHHHHH	251

248	YJ2.24	VKKLL	PQLICAFILR	AAQPAMAHHHHHH	252

249	8.3E	VKKLL	MVPLFPLPLP	AAQPAMAHHHHHH	253

250	8.1B	VKKLL	HqAILYYYLN	AAQPAMAHHHHHH	254

TABLE 3

Preference sequence patterns selected from
L3 S5 sc-dsFv library

			SEQ
			ID
No.	Code	Sequence	NO

	M13-pe1B	VKKLLFAIPL	VVPFYAAQPA	MAHHHHHH	4

1	2.1A	VKKLLFAIPL	LPAQAMPMSR	MAHHHHHH *	255

2	7.5C	VKKLLFAIPL	YFVLVRESSS	MAHHHHHH *	256

3	1.3B	VKKLLFAIPL	VLVVSSRTRA	MAHHHHHH *	257

4	YJ3.25	VKKLLFAIPL	LLSRPRAVPD	MAHHHHHH *	258

5	3.8A	VKKLLFAIPL	CVSVRSPAFA	MAHHHHHH *	259

6	1.6A	VKKLLFAIPL	MTTLASRTHA	MAHHHHHH *	260

7	1.4H	VKKLLFAIPL	YLSMTRSGAA	MAHHHHHH *	261

8	7.8F	VKKLLFAIPL	WLRSSVPVDS	MAHHHHHH *	262

9	7.8H	VKKLLFAIPL	LSSLTRDSSS	MAHHHHHH *	263

10	7.5E	VKKLLFAIPL	GLFTIRDSFA	MAHHHHHH *	264

11	7.6C	VKKLLFAIPL	WLGITKPVWS	MAHHHHHH *	265

12	1.3F	VKKLLFAIPL	YTLTPRPVFS	MAHHHHHH *	266

13	1.5F	VKKLLFAIPL	qLALSRPSFP	MAHHHHHH *	267

14	14.9A	VKKLLFAIPL	SSFLVADQSS	MAHHHHHH *	268

15	YJ3.7	VKKLLFAIPL	LLGLASPRSR	MAHHHHHH *	269

16	13.1E	VKKLLFAIPL	LTLSNRSAWS	MAHHHHHH *	270

17	2.2C	VKKLLFAIPL	LSLYPTRSTA	MAHHHHHH *	271

18	YJ3.10	VKKLLFAIPL	LTTLSRPSFS	MAHHHHHH *	272

19	8.1A	VKKLLFAIPL	YFSRPPqPSS	MAHHHHHH *	273

20	6.2H	VKKLLFAIPL	TMSSPPRSTS	MAHHHHHH *	274

21	8.1C	VKKLLFAIPL	YFLRISPSAS	MAHHHHHH *	275

22	1.8B	VKKLLFAIPL	LFLRPSAARP	MAHHHHHH *	276

23	1.8C	VKKLLFAIPL	LWSSSRPTSQ	MAHHHHHH *	277

24	YJ3.41	VKKLLFAIPL	YLVCSRPLHA	MAHHHHHH *	278

25	10.8G	VKKLLFAIPL	VLQRPPSPNT	MAHHHHHH *	279

26	2.7C	VKKLLFAIPL	AMASFRPRDQ	MAHHHHHH *	280

27	7.10C	VKKLLFAIPL	SRSLAMQPLP	MAHHHHHH *	281

28	1.2A	VKKLLFAIPL	LSSLRSSNPE	MAHHHHHH *	282

29	YJ3.4	VKKLLFAIPL	SILINFRASS	MAHHHHHH *	283

30	1.6B	VKKLLFAIPL	YWRSFWEPPA	MAHHHHHH *	284

31	4.8E	VKKLLFAIPL	YLAAPRSTVA	MAHHHHHH *	285

32	6.7H	VKKLLFAIPL	QYSAFSMSPR	MAHHHHHH *	286

33	7.9C	VKKLLFAIPL	YLVSSKNSYP	MAHHHHHH *	287

34	YJ3.72	VKKLLFAIPL	GLSVSFRTSA	MAHHHHHH *	288

35	4.4C	VKKLLFAIPL	AMLEPTRSSA	MAHHHHHH *	289

36	11.1B	VKKLLFAIPL	SLSLHRPALA	MAHHHHHH *	290

37	6.6B	VKKLLFAIPL	LSASARGSYA	MAHHHHHH *	291

38	YJ3.26	VKKLLFAIPL	YLAVTHRAYS	MAHHHHHH *	292

39	YJ3.44	VKKLLFAIPL	FFSLSRYSLA	MAHHHHHH *	293

40	5.4B	VKKLLFAIPL	YLSAPRHASP	MAHHHHHH *	294

41	5.2D	VKKLLFAIPL	WSFSRLPSSD	MAHHHHHH *	295

42	12.4E	VKKLLFAIPL	YLSLTKPSLS	MAHHHHHH *	296

43	14.1C	VKKLLFAIPL	SSPATEVLSP	MAHHHHHH *	297

44	6.2C	VKKLLFAIPL	TLFLQRSSLA	MAHHHHHH *	298

45	YJ3.6	VKKLLFAIPL	VFTRVPHKPS	MAHHHHHH *	299

46	4.1E	VKKLLFAIPL	AITRSSQFPS	MAHHHHHH *	300

47	6.4H	VKKLLFAIPL	LGDLRSSPDA	MAHHHHHH *	301

48	YJ3.53	VKKLLFAIPL	VTTLSTRCYA	MAHHHHHH *	302

49	7.7B	VKKLLFAIPL	FDASLEGPAM	MAHHHHHH *	303

50	11.3C	VKKLLFAIPL	YFSSPSSRAP	MAHHHHHH *	304

51	1.12A	VKKLLFAIPL	WFSFPFRSAA	MAHHHHHH	305

52	12.1A	VKKLLFAIPL	YLSMSSPARS	MAHHHHHH	306

53	1.12D	VKKLLFAIPL	SWSLCRPVCA	MAHHHHHH	307

54	4.3G	VKKLLFAIPL	LYCWPRHSWS	MAHHHHHH	308

55	YJ3.38	VKKLLFAIPL	IFYTTRSSLS	MAHHHHHH	309

56	YJ3.45	VKKLLFAIPL	IYTLRSHSMT	MAHHHHHH	400

57	2.9H	VKKLLFAIPL	PVPSLLGSAD	MAHHHHHH	401

58	9.5A	VKKLLFAIPL	SLSLNSRSYP	MAHHHHHH	402

59	2.7H	VKKLLFAIPL	FSPTSQEIRH	MAHHHHHH	403

60	2.2G	VKKLLFAIPL	YFSCPLRVAS	MAHHHHHH	404

61	YJ3.81	VKKLLFAIPL	VLSLNRGVFA	MAHHHHHH	405

62	7.4H	VKKLLFAIPL	SPqVLSSSPG	MAHHHHHH	406

63	4.2C	VKKLLFAIPL	YVNAMSSPRP	MAHHHHHH	407

64	13.6D	VKKLLFAIPL	YFTFVRSSWC	MAHHHHHH	408

65	5.8D	VKKLLFAIPL	FDLSSDSVSP	MAHHHHHH	409

66	YJ3.47	VKKLLFAIPL	YILFWRNTHA	MAHHHHHH	410

67	13.7A	VKKLLFAIPL	SCFLSRSAFS	MAHHHHHH	411

68	YJ3.83	VKKLLFAIPL	FFMITSKSRS	MAHHHHHH	412

69	12.6C	VKKLLFAIPL	IVSSSRGSFA	MAHHHHHH	413

70	4.10B	VKKLLFAIPL	AASRPLSPAA	MAHHHHHH	414

71	YJ3.46	VKKLLFAIPL	WLFSPLRSYS	MAHHHHHH	415

72	YJ3.56	VKKLLFAIPL	FLSYVRPLSA	MAHHHHHH	416

73	13.5G	VKKLLFAIPL	FIFTPRSVHS	MAHHHHHH	417

74	2.2E	VKKLLFAIPL	VSSIYKNSPP	MAHHHHHH	418

75	5.5H	VKKLLFAIPL	MSDSTAPSFA	MAHHHHHH	419

76	6.4B	VKKLLFAIPL	TLPqPRFPSP	MAHHHHHH	420

77	7.10G	VKKLLFAIPL	SLLADSPRRP	MAHHHHHH	421

78	5.3A	VKKLLFAIPL	FTDNSGEPSL	MAHHHHHH	422

79	11.1E	VKKLLFAIPL	YCMPMSRTCA	MAHHHHHH	423

80	11.1D	VKKLLFAIPL	MSRLSYHTPS	MAHHHHHH	424

81	2.2F	VKKLLFAIPL	LSNSRVPPSS	MAHHHHHH	425

82	15.7A	VKKLLFAIPL	FFASMRHTqA	MAHHHHHH	426

83	YJ3.5	VKKLLFAIPL	LLSTIKTSFS	MAHHHHHH	427

84	3.3A	VKKLLFAIPL	FQQSSLSSVP	MAHHHHHH	428

85	16.11A	VKKLLFAIPL	TLILSHRSSA	MAHHHHHH	429

86	11.12A	VKKLLFAIPL	SFSRDPSFTS	MAHHHHHH	430

87	9.1B	VKKLLFAIPL	ALSPTRHTLA	MAHHHHHH	431

88	13.9A	VKKLLFAIPL	NILFTVRVYA	MAHHHHHH	432

89	YJ3.15	VKKLLFAIPL	LASLSARCHG	MAHHHHHH	433

90	12.6B	VKKLLFAIPL	SVTLSLRASA	MAHHHHHH	434

91	15.8H	VKKLLFAIPL	SHDPLLLSSP	MAHHHHHH	435

92	YJ3.71	VKKLLFAIPL	LWSLSSRGMT	MAHHHHHH	436

93	YJ3.82	VKKLLFAIPL	LISYCRPVSS	MAHHHHHH	437

94	9.1D	VKKLLFAIPL	HSVELPASPA	MAHHHHHH	438

95	9.6A	VKKLLFAIPL	LLSTSRSSSG	MAHHHHHH	439

96	YJ3.34	VKKLLFAIPL	WFSCSRFALS	MAHHHHHH	440

97	YJ3.28	VKKLLFAIPL	VCTLSSRAFS	MAHHHHHH	441

98	11.1H	VKKLLFAIPL	YSPLARNPFS	MAHHHHHH	442

99	16.9D	VKKLLFAIPL	FFAFSRQSSG	MAHHHHHH	443

100	YJ3.70	VKKLLFAIPL	TFSIFSRALA	MAHHHHHH	444

101	YJ3.55	VKKLLFAIPL	SLFFSARAIA	MAHHHHHH	445

102	9.7A	VKKLLFAIPL	SQPSLCDPVP	MAHHHHHH	446

103	10.11A	VKKLLFAIPL	LASYHRVAFA	MAHHHHHH	447

104	10.1F	VKKLLFAIPL	WQLWQLPSRP	MAHHHHHH	448

105	16.8A	VKKLLFAIPL	FTPMYRPTSP	MAHHHHHH	449

106	YJ3.27	VKKLLFAIPL	LLSLHRFSFA	MAHHHHHH	450

107	9.5H	VKKLLFAIPL	SYSHPQNALA	MAHHHHHH	451

108	10.12D	VKKLLFAIPL	YVLRSDASWG	MAHHHHHH	452

109	4.2D	VKKLLFAIPL	FSGPPFDRTS	MAHHHHHH	453

110	YJ3.66	VKKLLFAIPL	FCALSRFTHA	MAHHHHHH	454

111	YJ3.24	VKKLLFAIPL	FSLSRPVPPL	MAHHHHHH	455

112	10.7D	VKKLLFAIPL	SMDSFSRPFF	MAHHHHHH	456

113	15.7C	VKKLLFAIPL	YTIIPSRASS	MAHHHHHH	457

114	15.12C	VKKLLFAIPL	VPSANPPPLS	MAHHHHHH	458

115	15.7E	VKKLLFAIPL	YLIKPPEGFS	MAHHHHHH	459

116	YJ3.42	VKKLLFAIPL	ISTLHFRAFG	MAHHHHHH	460

117	YJ3.37	VKKLLFAIPL	VRVMCGHSYA	MAHHHHHH	461

118	YJ3.67	VKKLLFAIPL	VLSLSRTFSG	MAHHHHHH	462

119	YJ3.75	VKKLLFAIPL	WCALSRQSMP	MAHHHHHH	463

120	YJ3.86	VKKLLFAIPL	YFWSLRVSWP	MAHHHHHH	464

121	YJ3.33	VKKLLFAIPL	YILSPRLPPP	MAHHHHHH	465

122	YJ3.22	VKKLLFAIPL	VVAAHRFSYA	MAHHHHHH	466

123	YJ3.62	VKKLLFAIPL	YVHLTSKAIP	MAHHHHHH	467

124	YJ3.59	VKKLLFAIPL	SLTLYRSGWS	MAHHHHHH	468

125	YJ3.18	VKKLLFAIPL	YYALSGRPVT	MAHHHHHH	469

126	YJ3.79	VKKLLFAIPL	MLSLMRQSAP	MAHHHHHH	470

TABLE 4

Preference sequence patterns selected from
L4 S5 sc-dsFv library

			SEQ
			ID
No.	Code	Sequence	NO

	M13-pe1B	VKKLLFAIPLVVPFY	AAQPAMAHHH	HHH	4

1	1.11A	VKKLLFAIPLVVPFY	ARPLTRIQTP	HHH *	471

2	9.3D	VKKLLFAIPLVVPFY	LTQLSRREPS	HHH *	472

3	1.6B	VKKLLFAIPLVVPFY	ARSLATSPSR	HHH *	473

4	14.5H	VKKLLFAIPLVVPFY	PARSYMLVRP	HHH *	474

5	12.2A	VKKLLFAIPLVVPFY	SRSYMLLSRP	HHH *	475

6	12.6H	VKKLLFAIPLVVPFY	TRSALAFFLP	HHH *	476

7	YJ4.13	VKKLLFAIPLVVPFY	SRGFTLPRLI	HHH *	477

8	YJ4.1	VKKLLFAIPLVVPFY	SSAFTRPIRP	HHH *	478

9	12.2E	VKKLLFAIPLVVPFY	TRYSHAFMLI	HHH *	479

10	6.10B	VKKLLFAIPLVVPFY	ARPMSMFRSD	HHH *	480

11	8.4D	VKKLLFAIPLVVPFY	ASSMSqYRQN	HHH *	481

12	5.9H	VKKLLFAIPLVVPFY	ARSYSRPPSI	HHH *	482

13	10.8A	VKKLLFAIPLVVPFY	ASSMSRLRPH	HHH *	483

14	YJ4.3	VKKLLFAIPLVVPFY	CRSLSRPMLV	HHH *	484

15	4.6C	VKKLLFAIPLVVPFY	SRSMSLHPTA	HHH *	485

16	CM11	VKKLLFAIPLVVPFY	TRSMTRLAPP	HHH *	486

17	9.8H	VKKLLFAIPLVVPFY	TRAMSVSHKT	HHH *	487

18	13.1F	VKKLLFAIPLVVPFY	LLAPKPSVKR	HHH *	488

19	9.7A	VKKLLFAIPLVVPFY	SRPAPALSRL	HHH *	489

20	15.9C	VKKLLFAIPLVVPFY	AKAMSARYQS	HHH *	490

21	CM18	VKKLLFAIPLVVPFY	FASQRSSPIR	HHH *	491

22	CM24	VKKLLFAIPLVVPFY	CLSFTSARFq	HHH *	492

23	12.1A	VKKLLFAIPLVVPFY	PSASSRLSPK	HHH *	493

24	2.10G	VKKLLFAIPLVVPFY	ARSYTRVPLA	HHH *	494

25	CM2	VKKLLFAIPLVVPFY	ARSLTFLPPR	HHH *	495

26	9.4C	VKKLLFAIPLVVPFY	TTRVNAFMLV	HHH *	496

27	11.11H	VKKLLFAIPLVVPFY	QAFRPVPVRN	HHH *	497

28	11.8H	VKKLLFAIPLVVPFY	TSGMSRLRSW	HHH *	498

29	1.12C	VKKLLFAIPLVVPFY	SRSPSQLSSR	HHH *	499

30	16.12H	VKKLLFAIPLVVPFY	AFSLSRTSSK	HHH *	500

31	3.11F	VKKLLFAIPLVVPFY	FHRVQQFSPA	HHH *	501

32	9.2B	VKKLLFAIPLVVPFY	LDSMLTFRRS	HHH *	502

33	CM40	VKKLLFAIPLVVPFY	CRSLTSPLRM	HHH *	503

34	15.5B	VKKLLFAIPLVVPFY	SRSASFLRPI	HHH *	504

35	9.2F	VKKLLFAIPLVVPFY	MTFqSNSPRG	HHH *	505

36	CM38	VKKLLFAIPLVVPFY	CRPMTLRqPV	HHH *	506

37	CM5	VKKLLFAIPLVVPFY	VRPMSRVIMS	HHH *	507

38	CM36	VKKLLFAIPLVVPFY	SYGFSRPFSK	HHH *	508

39	11.9G	VKKLLFAIPLVVPFY	TRSCFAFMLP	HHH *	509

40	6.8B	VKKLLFAIPLVVPFY	AFSGAFRQSQ	HHH *	510

41	16.6B	VKKLLFAIPLVVPFY	LRAGSFSAAP	HHH *	511

42	CM22	VKKLLFAIPLVVPFY	SHSMAPPSRR	HHH *	512

43	CM31	VKKLLFAIPLVVPFY	CRSGTFGNIG	HHH *	513

44	11.5F	VKKLLFAIPLVVPFY	ARSMASTPLA	HHH *	514

45	YJ4.2	VKKLLFAIPLVVPFY	VYPLAPRLRD	HHH *	515

46	6.10H	VKKLLFAIPLVVPFY	SLPWRRTPFQ	HHH *	516

47	10.3D	VKKLLFAIPLVVPFY	MRTPPLSqRI	HHH *	517

48	CM28	VKKLLFAIPLVVPFY	ARSLSSYNAV	HHH *	518

49	12.4D	VKKLLFAIPLVVPFY	VHALARKSQF	HHH *	519

50	CM25	VKKLLFAIPLVVPFY	SRSFSSPSIT	HHH	520

51	13.5A	VKKLLFAIPLVVPFY	CRALSKPLPP	HHH	521

52	12.6C	VKKLLFAIPLVVPFY	CRPSAPKMLL	HHH	522

53	CM16	VKKLLFAIPLVVPFY	SRSMSYFqPL	HHH	523

54	4.2C	VKKLLFAIPLVVPFY	TRSLSRSIPH	HHH	524

55	16.6C	VKKLLFAIPLVVPFY	SQLHqSPGNP	HHH	525

56	10.10A	VKKLLFAIPLVVPFY	TRAIARPPYT	HHH	526

57	10.11G	VKKLLFAIPLVVPFY	ARSLSTVRFP	HHH	527

58	CM8	VKKLLFAIPLVVPFY	TRAFSSPLSN	HHH	528

59	9.6D	VKKLLFAIPLVVPFY	NRTPTIqRDS	HHH	529

60	8.4B	VKKLLFAIPLVVPFY	ARAVSRTVPT	HHH	530

61	8.5E	VKKLLFAIPLVVPFY	AqSMAVPIST	HHH	531

62	13.2C	VKKLLFAIPLVVPFY	PqPSRGFMLI	HHH	532

63	CM10	VKKLLFAIPLVVPFY	TRSMVFPAKV	HHH	533

64	CM26	VKKLLFAIPLVVPFY	SRSMTLKGPE	HHH	534

65	CM17	VKKLLFAIPLVVPFY	AFPFSRQPNA	HHH	535

66	CM7	VKKLLFAIPLVVPFY	SRALTSISGM	HHH	536

67	CM6	VKKLLFAIPLVVPFY	CRGMSLNVTR	HHH	537

68	6.10C	VKKLLFAIPLVVPFY	SHWRTQRPPE	HHH	538

69	CM45	VKKLLFAIPLVVPFY	ARSFSSPPGP	HHH	539

70	13.1G	VKKLLFAIPLVVPFY	IFPIEASARR	HHH	540

71	CM39	VKKLLFAIPLVVPFY	ASSMALRPRV	HHH	541

72	YJ4.74	VKKLLFAIPLVVPFY	SRAFSSTPAM	HHH	542

73	1.7F	VKKLLFAIPLVVPFY	SRSMVLQGPT	HHH	543

74	YJ4.28	VKKLLFAIPLVVPFY	SRSMTSPPYI	HHH	544

75	10.3B	VKKLLFAIPLVVPFY	ANRPQSTKNI	HHH	545

76	YJ4.56	VKKLLFAIPLVVPFY	SRALTMTPSF	HHH	546

77	4.6H	VKKLLFAIPLVVPFY	PTRLFAFMLT	HHH	547

78	14.12A	VKKLLFAIPLVVPFY	SRAMSPIPRQ	HHH	548

79	CM29	VKKLLFAIPLVVPFY	ARSMGSMWQL	HHH	549

80	YJ4.42	VKKLLFAIPLVVPFY	SFSMTRSSPL	HHH	550

81	CM42	VKKLLFAIPLVVPFY	SFSFTRqPLP	HHH	551

82	YJ4.33	VKKLLFAIPLVVPFY	NRVPSPASQT	HHH	552

83	YJ4.23	VKKLLFAIPLVVPFY	SFSFSKPRFS	HHH	553

84	CM27	VKKLLFAIPLVVPFY	ARSLTQFSSV	HHH	554

85	YJ4.39	VKKLLFAIPLVVPFY	ARCFSSPVAL	HHH	555

86	11.3B	VKKLLFAIPLVVPFY	GASSWWLFPS	HHH	556

87	YJ4.84	VKKLLFAIPLVVPFY	TPPQQQALLS	HHH	557

88	14.1F	VKKLLFAIPLVVPFY	SRGFSMAFFP	HHH	558

89	CM33	VKKLLFAIPLVVPFY	SLAMSRPqAS	HHH	559

90	13.12C	VKKLLFAIPLVVPFY	TYALTTFqSV	HHH	560

91	YJ4.44	VKKLLFAIPLVVPFY	QHAFTRPFRV	HHH	561

92	CM30	VKKLLFAIPLVVPFY	SRAFSSPSGS	HHH	562

93	13.11G	VKKLLFAIPLVVPFY	TSALARSPRV	HHH	563

94	4.8B	VKKLLFAIPLVVPFY	CRAMSSPFRP	HHH	564

95	4.2B	VKKLLFAIPLVVPFY	STFARSFMLT	HHH	565

96	9.2D	VKKLLFAIPLVVPFY	FPLSSRAFML	HHH *	566

97	YJ4.71	VKKLLFAIPLVVPFY	SRSMSTSPIL	HHH	567

98	9.6H	VKKLLFAIPLVVPFY	SFGLqLPqPF	HHH	568

99	CM37	VKKLLFAIPLVVPFY	SRSMSLSSDL	HHH	569

100	16.3E	VKKLLFAIPLVVPFY	AFPLARRPIN	HHH	570

101	12.1B	VKKLLFAIPLVVPFY	TSCRAMTLPR	HHH	571

102	CM23	VKKLLFAIPLVVPFY	TYPFSRAGPP	HHH	572

103	YJ4.47	VKKLLFAIPLVVPFY	ANQQALPFQL	HHH	573

104	YJ4.38	VKKLLFAIPLVVPFY	GWSMSLRSHS	HHH	574

105	4.11H	VKKLLFAIPLVVPFY	SPQVVTRKDL	HHH	575

106	12.9G	VKKLLFAIPLVVPFY	LRNAHAMASA	HHH	576

107	CM44	VKKLLFAIPLVVPFY	SRSGSFNVTP	HHH	577

108	11.3E	VKKLLFAIPLVVPFY	SRPLSRVPVF	HHH	578

109	11.9F	VKKLLFAIPLVVPFY	SKRMPPPISq	HHH	579

110	CM34	VKKLLFAIPLVVPFY	TRSMSSLPSP	HHH	580

111	14.11D	VKKLLFAIPLVVPFY	CRSSSSIFPL	HHH	581

112	CM15	VKKLLFAIPLVVPFY	RSAHAMSIQT	HHH	582

113	10.1H	VKKLLFAIPLVVPFY	GYCFSARIIR	HHH	583

114	9.10A	VKKLLFAIPLVVPFY	PHLSPLqPQq	HHH	584

115	CM43	VKKLLFAIPLVVPFY	SFSFSRFPGL	HHH	585

116	YJ4.48	VKKLLFAIPLVVPFY	SSSMSLRPQF	HHH	586

117	11.11D	VKKLLFAIPLVVPFY	SSPRARPVPP	HHH	587

118	CM46	VKKLLFAIPLVVPFY	ARSLSALSPY	HHH	588

119	12.5C	VKKLLFAIPLVVPFY	PVRqLHTNLR	HHH	589

120	10.2F	VKKLLFAIPLVVPFY	PTTSTPYqSP	HHH	590

121	CM21	VKKLLFAIPLVVPFY	VNALTFLPSq	HHH	591

122	CM41	VKKLLFAIPLVVPFY	ARSLSSPLTL	HHH	592

123	YJ4.25	VKKLLFAIPLVVPFY	TRPPTVGLRQ	HHH	593

124	CM14	VKKLLFAIPLVVPFY	TRALSPMSWq	HHH	594

125	YJ4.6	VKKLLFAIPLVVPFY	VFPFSRPLLR	HHH	595

126	CM1	VKKLLFAIPLVVPFY	VPRCLSMSLG	HHH	596

127	YJ4.87	VKKLLFAIPLVVPFY	QQPSFHPISR	HHH	597

128	CM32	VKKLLFAIPLVVPFY	SKAFSSFqAS	HHH	598

129	10.6H	VKKLLFAIPLVVPFY	GYSMSqSGLT	HHH	599

130	YJ4.40	VKKLLFAIPLVVPFY	AQALTTRGLA	HHH	600

131	YJ4.26	VKKLLFAIPLVVPFY	VKSLTRPAFL	HHH	601

132	12.4F	VKKLLFAIPLVVPFY	AqSRLRVYPP	HHH	602

133	4.5B	VKKLLFAIPLVVPFY	PAIGFMLLRY	HHH	603

134	12.3D	VKKLLFAIPLVVPFY	SFGTLVRPRP	HHH	604

135	CM3	VKKLLFAIPLVVPFY	IRRPVDPVMP	HHH	605

136	YJ4.19	VKKLLFAIPLVVPFY	FPLRQTHRYP	HHH	606

137	13.2H	VKKLLFAIPLVVPFY	THSMQRPTGR	HHH	607

138	10.5D	VKKLLFAIPLVVPFY	RHTqLSSSTS	HHH	608

139	15.10D	VKKLLFAIPLVVPFY	SCGFSRLSKA	HHH	609

140	CM35	VKKLLFAIPLVVPFY	SRSFSQLPHI	HHH	610

141	YJ4.43	VKKLLFAIPLVVPFY	SSSMSQLRPF	HHH	611

142	10.2B	VKKLLFAIPLVVPFY	CRTTFALQSS	HHH	612

143	CM19	VKKLLFAIPLVVPFY	AQSMSIRHNN	HHH	613

144	11.4E	VKKLLFAIPLVVPFY	NSRFRTTPPS	HHH	614

145	CM20	VKKLLFAIPLVVPFY	SVSMSRYQLS	HHH	615

146	CM12	VKKLLFAIPLVVPFY	SSGASRLRIL	HHH	616

147	YJ4.81	VKKLLFAIPLVVPFY	CWSLSRPRLL	HHH	617

148	10.1C	VKKLLFAIPLVVPFY	TSRSTKLTPS	HHH	618

149	11.6D	VKKLLFAIPLVVPFY	SRVSVAFMLM	HHH	619

150	YJ4.72	VKKLLFAIPLVVPFY	CLGRSMAPGP	HHH	620

151	14.1A	VKKLLFAIPLVVPFY	FVHRRDSSSL	HHH	621

152	YJ4.24	VKKLLFAIPLVVPFY	SLGFSRLTSL	HHH	622

153	13.2B	VKKLLFAIPLVVPFY	ASALSRRVPq	HHH	623

154	11.6B	VKKLLFAIPLVVPFY	TYPASWPRLR	HHH	624

155	9.2G	VKKLLFAIPLVVPFY	SRVSLAVTPS	HHH	625

156	10.11B	VKKLLFAIPLVVPFY	NNPFSSLSqq	HHH	626

157	11.8D	VKKLLFAIPLVVPFY	RPLPRPFAGN	HHH	627

158	CM4	VKKLLFAIPLVVPFY	GFSMTQYLPq	HHH	628

159	YJ4.75	VKKLLFAIPLVVPFY	SSALSRSFYP	HHH	629

160	YJ4.61	VKKLLFAIPLVVPFY	TQQRCFAMHI	HHH	630

161	YJ4.85	VKKLLFAIPLVVPFY	IKHFYNSRPS	HHH	631

162	YJ4.51	VKKLLFAIPLVVPFY	FTRLPKESSP	HHH	632

163	9.6G	VKKLLFAIPLVVPFY	LPAQPRVTRT	HHH	633

164	CM13	VKKLLFAIPLVVPFY	LRSMTLNTST	HHH	634

165	YJ4.35	VKKLLFAIPLVVPFY	PDTFSYSSQD	HHH	635

166	YJ4.41	VKKLLFAIPLVVPFY	FRNPQLPSSA	HHH	636

167	YJ4.50	VKKLLFAIPLVVPFY	FRPDRTPPSS	HHH	637

168	9.8C	VKKLLFAIPLVVPFY	qSHTILPLPA	HHH	638

169	CM9	VKKLLFAIPLVVPFY	SSAFqPMVSS	HHH	639

170	9.7H	VKKLLFAIPLVVPFY	QSRRLPILPL	HHH	640

171	YJ4.31	VKKLLFAIPLVVPFY	GQAYLPAPQL	HHH	641

172	9.11B	VKKLLFAIPLVVPFY	TSRPRETLFL	HHH	642

173	9.3G	VKKLLFAIPLVVPFY	TAASVVRSRD	HHH	643

174	10.5F	VKKLLFAIPLVVPFY	VRGAAPKFSV	HHH	644

175	YJ4.14	VKKLLFAIPLVVPFY	FRHQPASVST	HHH	645

176	9.8B	VKKLLFAIPLVVPFY	PTNAIAFFLq	HHH	646

177	YJ4.59	VKKLLFAIPLVVPFY	LKSLRSDTPN	HHH	647

178	YJ4.22	VKKLLFAIPLVVPFY	IKRPLPLAPT	HHH	648

179	11.11F	VKKLLFAIPLVVPFY	ASSSKSRFML	HHH	649

180	YJ4.82	VKKLLFAIPLVVPFY	PWKPRLLPPQ	HHH	650

181	9.1H	VKKLLFAIPLVVPFY	SRGFMLTLRY	HHH	651

182	9.8E	VKKLLFAIPLVVPFY	CKARGIMPVF	HHH	652

183	YJ4.17	VKKLLFAIPLVVPFY	ASLPRLTSQS	HHH	653

184	11.2B	VKKLLFAIPLVVPFY	qSSAFSYMLS	HHH	654

185	10.7A	VKKLLFAIPLVVPFY	SFSSQRFLRP	HHH	655

186	9.7G	VKKLLFAIPLVVPFY	TSSNTSRRFP	HHH	656

187	11.10B	VKKLLFAIPLVVPFY	NqTAATAPPR	HHH	657

188	10.8G	VKKLLFAIPLVVPFY	GAPLSWRRSY	HHH	658

189	9.10D	VKKLLFAIPLVVPFY	CRSVWCIPRP	HHH	659

190	9.1C	VKKLLFAIPLVVPFY	AKACLRPLQT	HHH	660

191	9.6F	VKKLLFAIPLVVPFY	CLASSHRHRP	HHH	661

192	11.3H	VKKLLFAIPLVVPFY	LRADSLAPKS	HHH	662

193	9.9F	VKKLLFAIPLVVPFY	SVPQFSGRSR	HHH	663

194	YJ4.78	VKKLLFAIPLVVPFY	VYPARFPAKT	HHH	664

195	YJ4.21	VKKLLFAIPLVVPFY	NFMLRHPQTF	HHH	665

196	YJ4.32	VKKLLFAIPLVVPFY	YVPRFPPKSA	HHH	666

197	YJ4.86	VKKLLFAIPLVVPFY	LSPMSRTRYV	HHH	667

198	YJ4.66	VKKLLFAIPLVVPFY	TYPLTKPYRP	HHH	668

199	YJ4.83	VKKLLFAIPLVVPFY	SSYWSHRKPP	HHH	669

200	10.8C	VKKLLFAIPLVVPFY	SPRTFAFFLM	HHH	670

201	11.1A	VKKLLFAIPLVVPFY	LGPGIRKKPA	HHH	671

202	9.4E	VKKLLFAIPLVVPFY	TRLCVAKVAG	HHH	672

203	11.2E	VKKLLFAIPLVVPFY	RSLPASGASR	HHH	673

204	10.5E	VKKLLFAIPLVVPFY	ASPRVKSYSP	HHH	674

205	9.10F	VKKLLFAIPLVVPFY	PSRTFAFYLV	HHH	675

206	9.4H	VKKLLFAIPLVVPFY	qqEFAMAHHH	HHH	676

207	11.8B	VKKLLFAIPLVVPFY	PqSSKAFFLN	HHH	677

208	11.2F	VKKLLFAIPLVVPFY	VKALRGSYPT	HHH	678

209	11.7F	VKKLLFAIPLVVPFY	TqPSqVRYML	HHH	679

210	11.9C	VKKLLFAIPLVVPFY	SARGqHVRPP	HHH	680

211	10.11C	VKKLLFAIPLVVPFY	STRCPGFFLq	HHH	681

212	11.6E	VKKLLFAIPLVVPFY	CPSVFSRTPP	HHH	682

213	11.3A	VKKLLFAIPLVVPFY	DASSWRHFLS	HHH	683

Example 4

Production of sc-dsFv Against H5 of Influenza Virus and Microarray Test

As described above, scFvs (8a and 12a) and their disulfide forms (ds-8a and ds-12a, respectively) to various hemagglutins (HAs) from different serotypes of influenza virus were developed. As shown in FIG. 5, the results indicated that selected scFv phage clones against H5 of influenza virus could be introduced to sc-dsFv directly but had lower binding affinity as compared with original scFvs. These results also suggested that the binding affinity could be enhanced by sc-dsFv phage panning procedures with the signal sequences described above.
The 8aS5 protein could be concentrated to 6 mg/ml without precipitation. The array studies suggested that 4 ng/spot of ds-8a protein could detect ˜10⁷viruses in solution by using 40 nm fluorescence beads. In conclusion, the signal sequence derived from sc-dsFv phage production against VEGF from monoclonal antibody could be applied for sc-dsFv phage production against hemagglutinin from natural antibody repertoire. The binding affinity could be enhanced by sc-dsFv phage panning procedures to produce sc-dsFv with high binding capacity and better stability than scFv for further applications.

Example 5

Soluble Non-fusion sc-dsFv Expressed with Suppressor E. coli Strain

The signal sequences resulting in the successful expression of the displayed sc-dsFv on phage rescued from suppressor E. coli strain ER2738 were more likely to result in secretion of the soluble non-fusion anti-VEGF sc-dsFv in a culture medium. Signal sequence phage library L4 was selected for binding to immobilized VEGF and the VEGF-binding enriched phage variants were amplified for the next round of selection/amplification cycle. The selection/amplification cycle was repeated for four rounds. After each round of selection/amplification cycle, a random collection of 96 phage variants were picked from the amplified phage population. These phage variants were used to infect E. coli ER2738 and the soluble sc-dsFv was expressed in the overnight cultures, which were tested for binding to immobilized VEGF with ELISA.
These random collections of phage variants were also used to infect E. coli HB2151 for the same assay to determine the sc-dsFv secretion. The result showed that, with ER2738 as the host, 0%, 0%, 2%, and 14% of the phage variants from 1^st, 2^nd, 3^rd, and 4^thround of selection/amplification cycle respectively secreted functional sc-dsFv binding to VEGF with ELISA signal greater than OD_450nm>0.6. But this trend was not found in the experiment with E. coli strain HB2151. This result indicated that signal sequence alteration could restore the secretion of the soluble non-fusion sc-dsFv and that the search for the optimum signal sequences could be facilitated with phage-based selection/amplification cycles on signal sequence libraries. This conclusion is applicable only to the E. coli suppressor strain ER2738 as the bacteria host for the M13 phage.

Example 6

Interface Disulfide Bond Formation in the sc-dsFv

One measurement for the folding quality of the sc-dsFv is the extent of the interface disulfide bond formation in the sc-dsFv. This measurement was determined by the ratio of the sc-dsFv-VEGF binding ELISA signal after the fXa (bovine factor Xa) treatment over that before the fXa treatment. FXa cleaves substrate sequence -IEGR- in the linker peptide connecting the two variable domains in the sc-dsFv construct. If the interface disulfide bond was not formed in the sc-dsFv, the cleavage of the linker peptide would result in dissociation of the variable domains and abolishment of the affinity against VEGF. Hence the ratio reflects the percentage of interface disulfide bond formation in the sc-dsFv. This measurement was validated with the positive control (anti-VEGF scFv(fXa+)/M13pIII-pelB with -IEGR- in the linker peptide but without the interface disulfide bond) and the negative control (anti-VEGF scFv(fXa−)/M13pIII-pelB without both the fXa cutting site and the interface disulfide bond).
FIG. 6A compared the extent of the interface disulfide bond formation in the secreted soluble sc-dsFv with the disulfide bond formation in the sc-dsFv displayed on phage surface for the signal sequence variants from the L4 library. Strong correlation between the two measurement is evident (R²=0.508, p-value=0.000158). As shown in FIG. 6A, signal sequence optimization could improve the disulfide bond formation in the sc-dsFv from ˜0% up to 40% of the secreted sc-dsFv molecule.
Another folding quality of the sc-dsFv was determined by the ratio of the normalized sc-dsFv-VEGF binding ELISA signal over the normalized quantity of the secreted sc-dsFv determined by electrophoresis and Western blot analysis. FIG. 6B compared the extent of the interface disulfide bond formation in the secreted soluble sc-dsFv with the folding qualities derived from electrophoresis and ELISA measurements for the signal sequence variants from the L4 library. The positive correlation (R²=0.296, p-value=0.062) shown in FIG. 6B indicated that the interface disulfide bond formation enhanced the affinity for the sc-dsFv-VEGF interaction. The plot also indicated that the selected variants resulted in secreted sc-dsFv with up to more than 10-fold VEGF-binding signals per unit quantity of secreted sc-dsFv compared with the positive control scFv(fXa+)/M13pIII-pelB, indicating that the secreted sc-dsFv from these signal sequence variants folded into antibody-like structure substantially more effectively that the scFv construct. This is most likely due to the stabilizing interface disulfide bond that is formed in the sc-dsFv but is absent in the scFv construct.

Example 7

Correlation Between the Stability of sc-dsFv and the Extent of the Interface Disulfide Bond Formation in the sc-dsFv

The effect of interface disulfide bond in stabilizing the sc-dsFv structure was demonstrated in FIG. 7. Secreted sc-dsFv from representative variants selected from each of the three libraries were expressed and incubated at 37° C. for 12 days and the affinities of the sc-dsFv's against VEGF were measured along the course of incubation. FIG. 7A shows the VEGF-binding affinity plotted against the time course of incubation for each of the selected variants. The VEGF affinity for the control anti-VEGF scFv dropped rapidly in the first few days of incubation, while a few variants from L4 library resulted in stable secreted sc-dsFv that were even gaining affinities against VEGF compared with freshly prepared secreted protein, presumably due to the increasingly stabilized sc-dsFv with the formation of the interface disulfide bond. The correlation between the two measurements shown in FIG. 7B is strong (R²=0.867 p-value=0.023), indicating that the interface disulfide bond could be one of the most important factors in stabilizing the secreted sc-dsFv in the culture medium.
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.

Claims

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) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₀X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:1),

(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:2), or

(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:3),

each of X₁-X₁₀in (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) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:1),

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

10. An isolated nucleic acid, comprising

a first nucleotide sequence encoding a signal peptide, and

(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:596), in which X₁is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X₂is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X₃is A, F, G, L, M, P, Q, R, S, T, V, or W; X₄is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₅is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₆is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₈is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;

(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:597), in which X₁A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X₂is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₃is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₄is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₅is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₆is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X₇is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X₈is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X₉is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or

(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:598), in which X₁is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₅is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X₆is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X₈is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is 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

(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:597), in which X₁A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X₂is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₃is A, C, D, F, G, H, I, L, M, N, P, O, R, S, T, V, W, or Y; X₄is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₅is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₆is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X₇is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X₈is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X₉is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or

(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:598), in which X₁is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₅is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X₆is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X₈is A, E, F, G, H, I, K, L, M, N, P, O, R, S, T, V, W, or Y; X₉is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is 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.