# Patent

## Title
Phage displaying system expressing single chain antibody

## Abstract

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

## Background
RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 12/854,632, filed on Aug. 11, 2010, which claims priority to U.S. Provisional Application No. 61/232,819, filed on Aug. 11, 2009. The contents of both prior applications are incorporated by reference in their 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-X10 in (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-X10 in (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 faulting 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-X10 in (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:596), in which X1 is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X2 is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X3 is A, F, G, L, M, P, Q, R, S, T, V, or W; X4 is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X5 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X6 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X7 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X8 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X9 is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X10 is 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 X1 A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X2 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X3 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X4 is A, C, E, F, H, 1, K, L, M, N, P, Q, R, S, T, V, W, or Y; X5 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X6 is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X7 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X8 is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V, X9 is A, C, D, F, H, L, M, N, P, Q, R, S, T, V, W, or Y; and X10 is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or
(c) VKKLLFAIPLVVPFYX1X2X3X4X5X6X7X8X9X10HHHGH (SEQ ID NO:598), in which X1 is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X2 is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X3 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X4 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X5 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X6 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X9 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X10 is 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:596), in which X1 is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X2 is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X3 is A, F, G, L, M, P, Q, R, S, T, V, or W; X4 is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X5 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X6 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X7 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X8 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X9 is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X10 is 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 X1 A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X2 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X3 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X4 is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X5 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X6 is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X7 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X8 is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V, X9 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X10 is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or
(c) VKKLLFAIPLVVPFYX1X2X3X4X5X6X7X8X9X10HHHGH (SEQ ID NO:598), in which X1 is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X2 is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X3 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X4 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X5 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X6 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X7 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X9 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X10 is 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 4th round 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). Av1 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-X10 in (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-X10 in (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:596), in which X1 is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X2 is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X3 is A, F, G, L, M, P, Q, R, S, T, V, or W; X4 is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X5 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X6 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X7 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X8 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X9 is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X10 is 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 X1 is A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X2 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X3 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X4 is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X5 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X6 is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X7 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X8 is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X9 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X10 is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or
(c) VKKLLFAIPLVVPFYX1X2X3X4X5X6X7X8X9X10HHHGH (SEQ ID NO:598), in which X1 is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X2 is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X3 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X4 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X5 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X6 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X9 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X10 is 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 NOS: 5-593 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:




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            C
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        control
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The value of the sample (CV) is the average normalized signal from VEGF-coated membrane; that of the sample (C0) is the averaged normalized signal from the un-coated and un-blocked membrane. Similarly, those of the control (CV) and control (C0) 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: 5-593 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, 381-429 and 476 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: 5-593 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:596), in which X1 is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X2 is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X3 is A, F, G, L, M, P, Q, R, S, T, V, or W; X4 is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X5 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X6 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X7 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X8 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X9 is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X10 is 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 X1 A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X2 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X3 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X4 is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X5 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X6 is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X7 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X8 is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X9 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X10 is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or
(c) VKKLLFAIPLVVPFYX1X2X3X4X5X6X7X8X9X10HHHGH (SEQ ID NO:598), in which X1 is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X2 is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X3 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X4 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X5 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X6 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X9 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X10 is 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:594) and the pelB signal sequence of Pectobacterium wasabiae (MKYLLPTAAAGLLLLAAQPAMA) (SEQ ID NO:595). 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, 1011 colony-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 1011 cfu 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 12YT (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 (1011 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 (1011 cfu) 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 (OD600 nm=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 (1011 cfu/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 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 PB ST 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 III, 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 (10n1/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  ̃107 PFU/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- (SEQ ID NO:599), 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- (SEQ ID NO:599) site in the linker peptide (-(G)4SIEGRS(G)4S-) (SEQ ID NO:600), while the scFv(fXa−) construct had the conventional -(G)4S(G)4S(G)4S- (SEQ ID NO:601) 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










No.
Code
Sequence
SEQ ID NO:
















M13-pelB
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
LNGTESAqLF
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
HgAILYYYLN
AAQPAMAHHHHHH
254
















TABLE 3 







Preference sequence patterns selected from L3 S5 sc-dsFv library










No.
Code
Sequence
SEQ ID NO
















M13-pelB
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
7.60
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
YESRPPqPSS
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
310


 


57
2.9H
VKKLLFAIPL
PVPSLLGSAD
MAHHHHHH
311


 


58
9.5A
VKKLLFAIPL
SLSLNSRSYP
MAHHHHHH
312


 


59
2.7H
VKKLLFAIPL
FSPTSQEIRH
MAHHHHHH
313


 


60
2.2G
VKKLLFAIPL
YFSCPLRVAS
MAHHHHHH
314


 


61
YJ3.81
VKKLLFAIPL
VLSLNRGVFA
MAHHHHHH
315


 


62
7.4H
VKKLLFAIPL
SPqVLSSSPG
MAHHHHHH
316


 


63
4.2C
VKKLLFAIPL
YVNAMSSPRP
MAHHHHHH
317


 


64
13.6D
VKKLLFAIPL
YFTFVRSSWC
MAHHHHHH
318


 


65
5.8D
VKKLLFAIPL
FDLSSDSVSP
MAHHHHHH
319


 


66
YJ3.47
VKKLLFAIPL
YILFWRNTHA
MAHHHHHH
320


 


67
13.7A
VKKLLFAIPL
SCFLSRSAFS
MAHHHHHH
321


 


68
YJ3.83
VKKLLFAIPL
FFMITSKSRS
MAHHHHHH
322


 


69
12.6C
VKKLLFAIPL
IVSSSRGSFA
MAHHHHHH
323


 


70
4.10B
VKKLLFAIPL
AASRPLSPAA
MAHHHHHH
324


 


71
YJ3.46
VKKLLFAIPL
WLFSPLRSYS
MAHHHHHH
325


 


72
YJ3.56
VKKLLFAIPL
FLSYVRPLSA
MAHHHHHH
326


 


73
13.5G
VKKLLFAIPL
FIFTPRSVHS
MAHHHHHH
327


 


74
2.2E
VKKLLFAIPL
VSSIYKNSPP
MAHHHHHH
328


 


75
5.5H
VKKLLFAIPL
MSDSTAPSFA
MAHHHHHH
329


 


76
6.4B
VKKLLFAIPL
TLPqPRFPSP
MAHHHHHH
330


 


77
7.10G
VKKLLFAIPL
SLLADSPRRP
MAHHHHHH
331


 


78
5.3A
VKKLLFAIPL
FTDNSGEPSL
MAHHHHHH
332


 


79
11.1E
VKKLLFAIPL
YCMPMSRTCA
MAHHHHHH
333


 


80
11.1D
VKKLLFAIPL
MSRLSYHTPS
MAHHHHHH
334


 


81
2.2F
VKKLLFAIPL
LSNSRVPPSS
MAHHHHHH
335


 


82
15.7A
VKKLLFAIPL
FFASMRHTqA
MAHHHHHH
336


 


83
YJ3.5
VKKLLFAIPL
LLSTIKTSFS
MAHHHHHH
337


 


84
3.3A
VKKLLFAIPL
FQQSSLSSVP
MAHHHHHH
338


 


85
16.11A
VKKLLFAIPL
TLILSHRSSA
MAHHHHHH
339


 


86
11.12A
VKKLLFAIPL
SFSRDPSFTS
MAHHHHHH
340


 


87
9.1B
VKKLLFAIPL
ALSPTRHTLA
MAHHHHHH
341


 


88
13.9A
VKKLLFAIPL
NILFTVRVYA
MAHHHHHH
342


 


89
YJ3.15
VKKLLFAIPL
LASLSARCHG
MAHHHHHH
343


 


90
12.6B
VKKLLFAIPL
SVTLSLRASA
MAHHHHHH
344


 


91
15.8H
VKKLLFAIPL
SHDPLLLSSP
MAHHHHHH
345


 


92
YJ3.71
VKKLLFAIPL
LWSLSSRGMT
MAHHHHHH
346


 


93
YJ3.82
VKKLLFAIPL
LISYCRPVSS
MAHHHHHH
347


 


94
9.1D
VKKLLFAIPL
HSVELPASPA
MAHHHHHH
348


 


95
9.6A
VKKLLFAIPL
LLSTSRSSSG
MAHHHHHH
349


 


96
YJ3.34
VKKLLFAIPL
WFSCSRFALS
MAHHHHHH
350


 


97
YJ3.28
VKKLLFAIPL
VCTLSSRAFS
MAHHHHHH
351


 


98
11.1H
VKKLLFAIPL
YSPLARNPFS
MAHHHHHH
352


 


99
16.9D
VKKLLFAIPL
FFAFSRQSSG
MAHHHHHH
353


 


100
YJ3.70
VKKLLFAIPL
TFSIFSRALA
MAHHHHHH
354


 


101
YJ3.55
VKKLLFAIPL
SLFFSARAIA
MAHHHHHH
355


 


102
9.7A
VKKLLFAIPL
SQPSLCDPVP
MAHHHHHH
356


 


103
10.11A
VKKLLFAIPL
LASYHRVAFA
MAHHHHHH
357


 


104
10.1F
VKKLLFAIPL
WQLWQLPSRP
MAHHHHHH
358


 


105
16.8A
VKKLLFAIPL
FTPMYRPTSP
MAHHHHHH
359


 


106
YJ3.27
VKKLLFAIPL
LLSLHRFSFA
MAHHHHHH
360


 


107
9.5H
VKKLLFAIPL
SYSHPQNALA
MAHHHHHH
361


 


108
10.12D
VKKLLFAIPL
YVLRSDASWG
MAHHHHHH
362


 


109
4.2D
VKKLLFAIPL
FSGPPFDRTS
MAHHHHHH
363


 


110
YJ3.66
VKKLLFAIPL
FCALSRFTHA
MAHHHHHH
364


 


111
YJ3.24
VKKLLFAIPL
FSLSRPVPPL
MAHHHHHH
365


 


112
10.7D
VKKLLFAIPL
SMDSFSRPFF
MAHHHHHH
366


 


113
15.7C
VKKLLFAIPL
YTIIPSRASS
MAHHHHHH
367


 


114
15.12C
VKKLLFAIPL
VPSANPPPLS
MAHHHHHH
368


 


115
15.7E
VKKLLFAIPL
YLIKPPEGFS
MAHHHHHH
369


 


116
YJ3.42
VKKLLFAIPL
ISTLHFRAFG
MAHHHHHH
370


 


117
YJ3.37
VKKLLFAIPL
VRVMCGHSYA
MAHHHHHH
371


 


118
YJ3.67
VKKLLFAIPL
VLSLSRTFSG
MAHHHHHH
372


 


119
YJ3.75
VKKLLFAIPL
WCALSRQSMP
MAHHHHHH
373


 


120
YJ3.86
VKKLLFAIPL
YFWSLRVSWP
MAHHHHHH
374


 


121
YJ3.33
VKKLLFAIPL
YILSPRLPPP
MAHHHHHH
375


 


122
YJ3.22
VKKLLFAIPL
VVAAHRFSYA
MAHHHHHH
376


 


123
YJ3.62
VKKLLFAIPL
YVHLTSKAIP
MAHHHHHH
377


 


124
YJ3.59
VKKLLFAIPL
SLTLYRSGWS
MAHHHHHH
378


 


125
YJ3.18
VKKLLFAIPL
YYALSGRPVT
MAHHHHHH
379


 


126
YJ3.79
VKKLLFAIPL
MLSLMRQSAP
MAHHHHHH
380
















TABLE 4 







Preference sequence patterns selected from L4 S5 sc-dsFv library












No.
Code

Sequence

SEQ ID NO
















M13-pelB
VKKLLFAIPLVVPFY
AAQPAMAHHH
HHH
4


 


1
1.11A
VKKLLFAIPLVVPFY
ARPLTRIQTP
HHH *
381


 


2
9.3D
VKKLLFAIPLVVPFY
LTQLSRREPS
HHH *
382


 


3
1.6B
VKKLLFAIPLVVPFY
ARSLATSPSR
HHH *
383


 


4
14.5H
VKKLLFAIPLVVPFY
PARSYMLVRP
HHH *
384


 


5
12.2A
VKKLLFAIPLVVPFY
SRSYMLLSRP
HHH *
385


 


6
12.6H
VKKLLFAIPLVVPFY
TRSALAFFLP
HHH *
386


 


7
YJ4.13 
VKKLLFAIPLVVPFY
SRGFTLPRLI
HHH *
387


 


8
YJ4.1
VKKLLFAIPLVVPFY
SSAFTRPIRP
HHH *
388


 


9
12.2E
VKKLLFAIPLVVPFY
TRYSHAFMLI
HHH *
389


 


10
6.10B
VKKLLFAIPLVVPFY
ARPMSMFRSD
HHH *
390


 


11
8.4D
VKKLLFAIPLVVPFY
ASSMSqYRQN
HHH *
391


 


12
5.9H
VKKLLFAIPLVVPFY
ARSYSRPPSI
HHH *
392


 


13
10.8A
VKKLLFAIPLVVPFY
ASSMSRLRPH
HHH *
393


 


14
YJ4.3
VKKLLFAIPLVVPFY
CRSLSRPMLV
HHH *
394


 


15
4.6C
VKKLLFAIPLVVPFY
SRSMSLHPTA
HHH *
395


 


16
CM11
VKKLLFAIPLVVPFY
TRSMTRLAPP
HHH *
396


 


17
9.8H
VKKLLFAIPLVVPFY
TRAMSVSHKT
HHH *
397


 


18
13.1F
VKKLLFAIPLVVPFY
LLAPKPSVKR
HHH *
398


 


19
9.7A
VKKLLFAIPLVVPFY
SRPAPALSRL
HHH *
399


 


20
15.9C
VKKLLFAIPLVVPFY
AKAMSARYQS
HHH *
400


 


21
CM18
VKKLLFAIPLVVPFY
FASQRSSPIR
HHH *
401


 


22
CM24
VKKLLFAIPLVVPFY
CLSFTSARFq
HHH *
402


 


23
12.1A
VKKLLFAIPLVVPFY
PSASSRLSPK
HHH *
403


 


24
2.10G
VKKLLFAIPLVVPFY
ARSYTRVPLA
HHH *
404


 


25
CM2
VKKLLFAIPLVVPFY
ARSLTFLPPR
HHH *
405


 


26
9.4C
VKKLLFAIPLVVPFY
TTRVNAFMLV
HHH *
406


 


27
11.11H
VKKLLFAIPLVVPFY
QAFRPVPVRN
HHH *
407


 


28
11.8H
VKKLLFAIPLVVPFY
TSGMSRLRSW
HHH *
408


 


29
1.12C
VKKLLFAIPLVVPFY
SRSPSQLSSR
HHH *
409


 


30
16.12H
VKKLLFAIPLVVPFY
AFSLSRTSSK
HHH *
410


 


31
3.11F
VKKLLFAIPLVVPFY
FHRVQQFSPA
HHH *
411


 


32
9.2B
VKKLLFAIPLVVPFY
LDSMLTFRRS
HHH *
412


 


33
CM40
VKKLLFAIPLVVPFY
CRSLTSPLRM
HHH *
413


 


34
15.5B
VKKLLFAIPLVVPFY
SRSASFLRPI
HHH *
414


 


35
9.2F
VKKLLFAIPLVVPFY
MTFqSNSPRG
HHH *
415


 


36
CM38
VKKLLFAIPLVVPFY
CRPMTLRqPV
HHH *
416


 


37
CM5
VKKLLFAIPLVVPFY
VRPMSRVIMS
HHH *
417


 


38
CM36
VKKLLFAIPLVVPFY
SYGFSRPFSK
HHH *
418


 


39
11.9G
VKKLLFAIPLVVPFY
TRSCFAFMLP
HHH *
419


 


40
6.8B
VKKLLFAIPLVVPFY
AFSGAFRQSQ
HHH *
420


 


41
16.6B
VKKLLFAIPLVVPFY
LRAGSFSAAP
HHH *
421


 


42
CM22
VKKLLFAIPLVVPFY
SHSMAPPSRR
HHH *
422


 


43
CM31
VKKLLFAIPLVVPFY
CRSGTFGNIG
HHH *
423


 


44
11.5F
VKKLLFAIPLVVPFY
ARSMASTPLA
HHH *
424


 


45
YJ4.2
VKKLLFAIPLVVPFY
VYPLAPRLRD
HHH *
425


 


46
6.10H
VKKLLFAIPLVVPFY
SLPWRRTPFQ
HHH *
426


 


47
10.3D
VKKLLFAIPLVVPFY
MRTPPLSqRI
HHH *
427


 


48
CM28
VKKLLFAIPLVVPFY
ARSLSSYNAV
HHH *
428


 


49
12.4D
VKKLLFAIPLVVPFY
VHALARKSQF
HHH *
429


 


50
CM25
VKKLLFAIPLVVPFY
SRSFSSPSIT
HHH
430


 


51
13.5A
VKKLLFAIPLVVPFY
CRALSKPLPP
HMH
431


 


52
12.6C
VKKLLFAIPLVVPFY
CRPSAPKMLL
HHH
432


 


53
CM16
VKKLLFAIPLVVPFY
SRSMSYFqPL
HHH
433


 


54
4.2C
VKKLLFAIPLVVPFY
TRSLSRSIPH
HHH
434


 


55
16.6C
VKKLLFAIPLVVPFY
SQLHqSPGNP
HHH
435


 


56
10.10A
VKKLLFAIPLVVPFY
TRAIARPPYT
HHH
436


 


57
10.11G
VKKLLFAIPLVVPFY
ARSLSTVRFP
HHH
437


 


58
CM8
VKKLLFAIPLVVPFY
TRAFSSPLSN
HHH
438


 


59
9.6D
VKKLLFAIPLVVPFY
NRTPTIqRDS
HHH
439


 


60
8.4B
VKKLLFAIPLVVPFY
ARAVSRTVPT
HHH
440


 


61
8.5E
VKKLLFAIPLVVPFY
AqSMAVPIST
HHH
441


 


62
13.2C
VKKLLFAIPLVVPFY
PqPSRGFMLI
HHH
442


 


63
CM10
VKKLLFAIPLVVPFY
TRSMVFPAKV
HHH
443


 


64
CM26
VKKLLFAIPLVVPFY
SRSMTLKGPE
HHH
444


 


65
CM17
VKKLLFAIPLVVPFY
AFPFSRQPNA
HHH
445


 


66
CM7
VKKLLFAIPLVVPFY
SRALTSISGM
HHH
446


 


67
CM6
VKKLLFAIPLVVPFY
CRGMSLNVTR
HHH
447


 


68
6.10C
VKKLLFAIPLVVPFY
SHWRTQRPPE
HHH
448


 


69
CM45
VKKLLFAIPLVVPFY
ARSFSSPPGP
HHH
449


 


70
13.1G
VKKLLFAIPLVVPFY
IFPIEASARR
HHH
450


 


71
CM39
VKKLLFAIPLVVPFY
ASSMALRPRV
HHH
451


 


72
YJ4.74
VKKLLFAIPLVVPFY
SRAFSSTPAM
HHH
452


 


73
1.7F
VKKLLFAIPLVVPFY
SRSMVLQGPT
HHH
453


 


74
YJ4.28
VKKLLFAIPLVVPFY
SRSMTSPPYI
HHH
454


 


75
10.3B
VKKLLFAIPLVVPFY
ANRPQSTKNI
HHH
455


 


76
YJ4.56
VKKLLFAIPLVVPFY
SRALTMTPSF
HHH
456


 


77
4.6H
VKKLLFAIPLVVPFY
PTRLFAFMLT
HHH
457


 


78
14.12A
VKKLLFAIPLVVPFY
SRAMSPIPRQ
HHH
458


 


79
CM29
VKKLLFAIPLVVPFY
ARSMGSMWQL
HHH
459


 


80
YJ4.42
VKKLLFAIPLVVPFY
SFSMTRSSPL
HHH
460


 


81
CM42
VKKLLFAIPLVVPFY
SFSFTRqPLP
HHH
461


 


82
YJ4.33
VKKLLFAIPLVVPFY
NRVPSPASQT
HHH
462


 


83
YJ4.23
VKKLLFAIPLVVPFY
SFSFSKPRFS
HHH
463


 


84
CM27
VKKLLFAIPLVVPFY
ARSLTQFSSV
HHH
464


 


85
YJ4.39
VKKLLFAIPLVVPFY
ARCFSSPVAL
HHH
465


 


86
11.3B
VKKLLFAIPLVVPFY
GASSWWLFPS
HHH
466


 


87
YJ4.84
VKKLLFAIPLVVPFY
TPPQQQALLS
HHH
467


 


88
14.1F
VKKLLFAIPLVVPFY
SRGFSMAFFP
HHH
468


 


89
CM33
VKKLLFAIPLVVPFY
SLAMSRPqAS
HHH
469


 


90
13.12C
VKKLLFAIPLVVPFY
TYALTTFqSV
HHH
470


 


91
YJ4.44
VKKLLFAIPLVVPFY
QHAFTRPFRV
HHH
471


 


92
CM30
VKKLLFAIPLVVPFY
SRAFSSPSGS
HHH
472


 


93
13.11G
VKKLLFAIPLVVPFY
TSALARSPRV
HHH
473


 


94
4.8E
VKKLLFAIPLVVPFY
CRAMSSPFRP
HHH
474


 


95
4.2B
VKKLLFAIPLVVPFY
STFARSFMLT
HHH
475


 


96
9.2D
VKKLLFAIPLVVPFY
FPLSSRAFML
HHH *
476


 


97
YJ4.71
VKKLLFAIPLVVPFY
SRSMSTSPIL
HHH
477


 


98
9.6H
VKKLLFAIPLVVPFY
SFGLqLPqPF
HHH
478


 


99
CM37
VKKLLFAIPLVVPFY
SRSMSLSSDL
HHH
479


 


100
16.3E
VKKLLFAIPLVVPFY
AFPLARRPIN
HHH
480


 


101
12.1B
VKKLLFAIPLVVPFY
TSCRAMTLPR
HHH
481


 


102
CM23
VKKLLFAIPLVVPFY
TYPFSRAGPP
HHH
482


 


103
YJ4.47
VKKLLFAIPLVVPFY
ANQQALPFQL
HHH
483


 


104
YJ4.38
VKKLLFAIPLVVPFY
GWSMSLRSHS
HHH
484


 


105
4.11H
VKKLLFAIPLVVPFY
SPQVVTRKDL
HHH
485


 


106
12.9G
VKKLLFAIPLVVPFY
LRNAHAMASA
HHH
486


 


107
CM44
VKKLLFAIPLVVPFY
SRSGSFNVTP
HHH
487


 


108
11.3E
VKKLLFAIPLVVPFY
SRPLSRVPVF
HHH
488


 


109
11.9F
VKKLLFAIPLVVPFY
SKRMPPPISq
HHH
489


 


110
CM34
VKKLLFAIPLVVPFY
TRSMSSLPSP
HHH
490


 


111
14.11DV
KKLLFAIPLVVPFY
CRSSSSIFPL
HHH
491


 


112
CM15
VKKLLFAIPLVVPFY
RSAHAMSIQT
HHH
492


 


113
10.1H
VKKLLFAIPLVVPFY
GYCFSARIIR
HHH
493


 


114
9.10A
VKKLLFAIPLVVPFY
PHLSPLqPQq
HHH
494


 


115
CM43
VKKLLFAIPLVVPFY
SFSFSRFPGL
HHH
495


 


116
YJ4.48
VKKLLFAIPLVVPFY
SSSMSLRPQF
HHH
496


 


117
11.11DV
KKLLFAIPLVVPFY
SSPRARPVPP
HHH
497


 


118
CM46
VKKLLFAIPLVVPFY
ARSLSALSPY
HHH
498


 


119
12.5C
VKKLLFAIPLVVPFY
PVRqLHTNLR
HHH
499


 


120
10.2F
VKKLLFAIPLVVPFY
PITSTPYqSP
HHH
500


 


121
CM21
VKKLLFAIPLVVPFY
VNALTFLPSq
HHH
501


 


122
CM41
VKKLLFAIPLVVPFY
ARSLSSPLTL
HHH
502


 


123
YJ4.25
VKKLLFAIPLVVPFY
TRPPTVGLRQ
HHH
503


 


124
CM14
VKKLLFAIPLVVPFY
TRALSPMSWq
HHH
504


 


125
YJ4.6
VKKLLFAIPLVVPFY
VFPFSRPLLR
HHH
505


 


126
CM1
VKKLLFAIPLVVPFY
VPRCLSMSLG
HHH
506


 


127
YJ4.87
VKKLLFAIPLVVPFY
QQPSFHPISR
HHH
507


 


128
CM32
VKKLLFAIPLVVPFY
SKAFSSFqAS
HHH
508


 


129
10.6H
VKKLLFAIPLVVPFY
GYSMSqSGLT
HHH
509


 


130
YJ4.40
VKKLLFAIPLVVPFY
AQALTTRGLA
HHH
510


 


131
YJ4.26
VKKLLFAIPLVVPFY
VKSLTRPAFL
HHH
511


 


132
12.4F
VKKLLFAIPLVVPFY
AqSRLRVYPP
HHH
512


 


133
4.5B
VKKLLFAIPLVVPFY
PAIGFMLLRY
HHH
513


 


134
12.3D
VKKLLFAIPLVVPFY
SFGTLVRPRP
HHH
514


 


135
CM3
VKKLLFAIPLVVPFY
IRRPVDPVMP
HHH
515


 


136
YJ4.19
VKKLLFAIPLVVPFY
FPLRQTHRYP
HHH
516


 


137
13.2H
VKKLLFAIPLVVPFY
THSMQRPTGR
HHH
517


 


138
10.5D
VKKLLFAIPLVVPFY
RHTqLSSSTS
HHH
518


 


139
15.10D
VKKLLFAIPLVVPFY
SCGFSRLSKA
HHH
519


 


140
CM35
VKKLLFAIPLVVPFY
SRSFSQLPHI
HHH
520


 


141
YJ4.43
VKKLLFAIPLVVPFY
SSSMSQLRPF
HHH
521


 


142
10.2B
VKKLLFAIPLVVPFY
CRTTFALQSS
HHH
522


 


143
CM19
VKKLLFAIPLVVPFY
AQSMSIRHNN
HHH
523


 


144
11.4E
VKKLLFAIPLVVPFY
NSRFRTTPPS
HHH
524


 


145
CM20
VKKLLFAIPLVVPFY
SVSMSRYQLS
HHH
525


 


146
CM12
VKKLLFAIPLVVPFY
SSGASRLRIL
HHH
526


 


147
YJ4.81
VKKLLFAIPLVVPFY
CWSLSRPRLL
HHH
527


 


148
10.1C
VKKLLFAIPLVVPFY
TSRSTKLTPS
HHH
528


 


149
11.6D
VKKLLFAIPLVVPFY
SRVSVAFMLM
HHH
529


 


150
YJ4.72
VKKLLFAIPLVVPFY
CLGRSMAPGP
HHH
530


 


151
14.1A
VKKLLFAIPLVVPFY
FVHRRDSSSL
HHH
531


 


152
YJ4.24
VKKLLFAIPLVVPFY
SLGFSRLTSL
HHH
532


 


153
13.2B
VKKLLFAIPLVVPFY
ASALSRRVPq
HHH
533


 


154
11.6B
VKKLLFAIPLVVPFY
TYPASWPRLR
HHH
534


 


155
9.2G
VKKLLFAIPLVVPFY
SRVSLAVTPS
HHH
535


 


156
10.11B
VKKLLFAIPLVVPFY
NNPFSSISqq
HHH
536


 


157
11.8D
VKKLLFAIPLVVPFY
RPLPRPFAGN
HHH
537


 


158
CM4
VKKLLFAIPLVVPFY
GFSMTQYLPq
HHH
538


 


159
YJ4.75
VKKLLFAIPLVVPFY
SSALSRSFYP
HHH
539


 


160
YJ4.61
VKKLLFAIPLVVPFY
TQQRCFAMHI
HHH
540


 


161
YJ4.85
VKKLLFAIPLVVPFY
IKHFYNSRPS
HHH
541


 


162
YJ4.51
VKKLLFAIPLVVPFY
FTRLPKESSP
HHH
542


 


163
9.6G
VKKLLFAIPLVVPFY
LPAQPRVTRT
HHH
543


 


164
CM13
VKKLLFAIPLVVPFY
LRSMTLNTST
HHH
544


 


165
YJ4.35
VKKLLFAIPLVVPFY
PDTFSYSSQD
HHH
545


 


166
YJ4.41
VKKLLFAIPLVVPFY
FRNPQLPSSA
HHH
546


 


167
YJ4.50
VKKLLFAIPLVVPFY
FRPDRTPPSS
HHH
547


 


168
9.8C
VKKLLFAIPLVVPFY
qSHTILPLPA
HHH
548


 


169
CM9
VKKLLFAIPLVVPFY
SSAFqPMVSS
HHH
549


 


170
9.7H
VKKLLFAIPLVVPFY
QSRRLPILPL
HHH
550


 


171
YJ4.31
VKKLLFAIPLVVPFY
GQAYLPAPQL
HHH
551


 


172
9.11B
VKKLLFAIPLVVPFY
TSRPRETLFL
HHH
552


 


173
9.3G
VKKLLFAIPLVVPFY
TAASVVRSRD
HHH
553


 


174
10.5F
VKKLLFAIPLVVPFY
VRGAAPKFSV
HHH
554


 


175
YJ4.14
VKKLLFAIPLVVPFY
FRHQPASVST
HHH
555


 


176
9.8B
VKKLLFAIPLVVPFY
PTNAIAFFLq
HHH
556


 


177
YJ4.59
VKKLLFAIPLVVPFY
LKSLRSDTPN
HHH
557


 


178
YJ4.22
VKKLLFAIPLVVPFY
IKRPLPLAPT
HHH
558


 


179
11.11F
VKKLLFAIPLVVPFY
ASSSKSRFML
HHH
559


 


180
YJ4.82
VKKLLFAIPLVVPFY
PWKPRLLPPQ
HHH
560


 


181
9.1H
VKKLLFAIPLVVPFY
SRGFMLTLRY
HHH
561


 


182
9.8E
VKKLLFAIPLVVPFY
CKARGIMPVF
HHH
562


 


183
YJ4.17
VKKLLFAIPLVVPFY
ASLPRLTSQS
HHH
563


 


184
11.2B
VKKLLFAIPLVVPFY
qSSAFSYMLS
HHH
564


 


185
10.7A
VKKLLFAIPLVVPFY
SFSSQRFLRP
HHH
565


 


186
9.7G
VKKLLFAIPLVVPFY
TSSNTSRRFP
HHH
566


 


187
11.10B
VKKLLFAIPLVVPFY
NqTAATAPPR
HHH
567


 


188
10.8G
VKKLLFAIPLVVPFY
GAPLSWRRSY
HHH
568


 


189
9.10D
VKKLLFAIPLVVPFY
CRSVWCIPRP
HHH
569


 


190
9.1C
VKKLLFAIPLVVPFY
AKACLRPLQT
HHH
570


 


191
9.6F
VKKLLFAIPLVVPFY
CLASSHRHRP
HHH
571


 


192
11.3H
VKKLLFAIPLVVPFY
LRADSLAPKS
HHH
572


 


193
9.9F
VKKLLFAIPLVVPFY
SVPQFSGRSR
HHH
573


 


194
YJ4.78
VKKLLFAIPLVVPFY
VYPARFPAKT
HHH
574


 


195
YJ4.21
VKKLLFAIPLVVPFY
NFMLRHPQTF
HHH
575


 


196
YJ4.32
VKKLLFAIPLVVPFY
YVPRFPPKSA
HHH
576


 


197
YJ4.86
VKKLLFAIPLVVPFY
LSPMSRTRYV
HHH
577


 


198
YJ4.66
VKKLLFAIPLVVPFY
TYPLTKPYRP
HHH
578


 


199
YJ4.83
VKKLLFAIPLVVPFY
SSYWSHRKPP
HHH
579


 


200
10.8C
VKKLLFAIPLVVPFY
SPRTFAFFLM
HHH
580


 


201
11.1A
VKKLLFAIPLVVPFY
LGPGIRKKPA
HHH
581


 


202
9.4E
VKKLLFAIPLVVPFY
TRLCVAKVAG
HHH
582


 


203
11.2E
VKKLLFAIPLVVPFY
RSLPASGASR
HHH
583


 


204
10.5E
VKKLLFAIPLVVPFY
ASPRVKSYSP
HHH
584


 


205
9.10F
VKKLLFAIPLVVPFY
PSRTFAFYLV
HHH
585


 


206
9.4H
VKKLLFAIPLVVPFY
qqEFAMAHHH
HHH
586


 


207
11.8B
VKKLLFAIPLVVPFY
PqSSKAFFLN
HHH
587


 


208
11.2F
VKKLLFAIPLVVPFY
VKALRGSYPT
HHH
588


 


209
11.7F
VKKLLFAIPLVVPFY
TqPSqVRYML
HHH
589


 


210
11.9C
VKKLLFAIPLVVPFY
SARGqHVRPP
HHH
590


 


211
10.11C
VKKLLFAIPLVVPFY
STRCPGFFLq
HHH
591


 


212
11.6E
VKKLLFAIPLVVPFY
CPSVFSRTPP
HHH
592


 


213 
11.3A
VKKLLFAIPLVVPFY
DASSWRHFLS
HHH
593









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  ̃107 viruses 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 1st, 2nd, 3rd, and 4th round of selection/amplification cycle respectively secreted functional sc-dsFv binding to VEGF with ELISA signal greater than OD450 nm>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- (SEQ ID NO:599) 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. An isolated nucleic acid, comprising
a first nucleotide sequence encoding a signal peptide, and
a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3′ downstream to the first nucleotide, wherein the signal peptide has the amino acid sequence of
VKKLLX1X2X3X4X5X6X7X8X9X10AAQPAMAHHHHHHGH (SEQ ID NO:596), in which X1 is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X2 is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X3 is A, F, G, L, M, P, Q, R, S, T, V, or W; X4 is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X5 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X6 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X7 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X8 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X9 is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X10 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y.

2. The nucleic acid of claim 1, further comprising 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 of claim 1, wherein the nucleic acid is an expression vector for expression a fusion protein containing the signal peptide and the single chain antibody.

4. The nucleic acid 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 (VH) or a light chain variable region (VL).

6. The nucleic acid library of claim 4, wherein the second variable region is a heavy chain variable region (VH) or a light chain variable region (VL).

7. The nucleic acid of claim 3, wherein the expression vector is a phagemid.

8. A host cell containing the nucleic acid of claim 4.

9. 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 8, and
culturing the host cell in a medium under conditions allowing expression of the phage.

10. 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
VKKLLX1X2X3X4X5X6X7X8X9X10AAQPAMAHHHHHHGH (SEQ ID NO:596), in which X1 is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X2 is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X3 is A, F, G, L, M, P, Q, R, S, T, V, or W; X4 is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X5 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X6 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X7 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X8 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X9 is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X10 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y.

11. The method 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.

12. The method of claim 11, wherein the first variable region is a heavy chain variable region (VH) or a light chain variable region (VL).

13. The method of claim 11, wherein the second variable region is a heavy chain variable region (VH) or a light chain variable region (VL).

14. The method of claim 10, further comprising, after the culturing step, collecting the medium for isolating the disulfide-stabilized single chain antibody.

15. The method of claim 10, 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.

16. The method of claim 15, further comprising, after the culturing step, collecting the medium for isolating phage particles that display the disulfide-stabilized single chain antibody.