Easy To Use Patents Search & Patent Lawyer Directory

At Patents you can conduct a Patent Search, File a Patent Application, find a Patent Attorney, or search available technology through our Patent Exchange. Patents are available using simple keyword or date criteria. If you are looking to hire a patent attorney, you've come to the right place. Protect your idea and hire a patent lawyer.


Search All Patents:



  This Patent May Be For Sale or Lease. Contact Us

  Is This Your Patent? Claim This Patent Now.



Register or Login To Download This Patent As A PDF




United States Patent Application 20180037639
Kind Code A1
Crowe; Scott ;   et al. February 8, 2018

PEPTIDE CONSTRUCT HAVING A PROTEASE-CLEAVABLE LINKER

Abstract

There is provided inter alia a construct suitable for oral administration comprising a first polypeptide and a second polypeptide connected by a labile peptide linker, wherein the labile peptide linker is labile to one or more proteases present in the intestinal tract and wherein the first and second polypeptides are substantially resistant to said one or more proteases.


Inventors: Crowe; Scott; (Cambridge, GB) ; West; Mike; (Cambridge, GB) ; Roberts; Kevin; (Cambridge, GB)
Applicant:
Name City State Country Type

VHSQUARED LIMITED

Cambridge

GB
Family ID: 1000002960000
Appl. No.: 15/717174
Filed: September 27, 2017


Related U.S. Patent Documents

Application NumberFiling DatePatent Number
PCT/EP2016/057022Mar 31, 2016
15717174

Current U.S. Class: 1/1
Current CPC Class: C07K 16/1282 20130101; C07K 16/241 20130101; C07K 2319/50 20130101; C07K 2317/56 20130101; C07K 2317/94 20130101
International Class: C07K 16/12 20060101 C07K016/12; C07K 16/24 20060101 C07K016/24

Foreign Application Data

DateCodeApplication Number
Mar 31, 2015EP15162112.5
Mar 31, 2015EP15162114.1
Mar 31, 2015EP15162115.8
Jan 21, 2016EP16152320.4

Claims



1. A method of making a construct comprising a first immunoglobulin chain variable domain and a second immunoglobulin chain variable domain connected by a labile peptide linker, wherein: (i) the peptide linker is labile to one or more proteases present in the intestinal tract, (ii) the peptide linker is stable to yeast proteases and (iii) the first and second immunoglobulin chain variable domains are substantially resistant to said one or more proteases; comprising the step of producing the construct in yeast.

2. The method according to claim 1, wherein the yeast is S. cerevisiae.

3. The method according claim 1, wherein the labile peptide linker is labile to one or more proteases present in the intestinal tract such that greater than 50% by mass of the construct is cleaved into first and second immunoglobulin chain variable domains after 160 minutes after mixing in the Trypsin Protease Assay.

4. The method according to claim 1, wherein the labile peptide linker is stable to yeast proteases such that no more than 10% by mass of the construct is cleaved into first and second immunoglobulin chain variable domains after producing the construct using the Yeast Expression Protocol.

5. The method according to claim 1, wherein the first immunoglobulin chain variable domain and the second immunoglobulin chain variable domain are substantially resistant to one or more proteases present in the intestinal tract such that at least 70% by mass of the first immunoglobulin chain variable domain and at least 70% by mass of the second immunoglobulin chain variable domain remain uncleaved after 10 minutes after mixing in the Trypsin Protease Assay.

6. The method according to claim 1, wherein the one or more proteases are present in the small or large intestine.

7. The method according to claim 1, wherein the labile peptide linker comprises a cleavage site for trypsin or a trypsin-like protease.

8. The method according to claim 1, wherein the labile peptide linker comprises at least 1 K residue.

9. The method according to claim 1, wherein the labile peptide linker comprises at least 1 R residue.

10. The method according to claim 1, wherein the labile peptide linker has a length of at least 3 residues.

11. The method according to claim 1, wherein the labile peptide linker has a length of no greater than 40 residues.

12. The method according to claim 1, wherein the labile peptide linker comprises of a polypeptide sequence of the format: [-(G.sub.aS).sub.x--B-(G.sub.bS).sub.y--].sub.z wherein a is 1 to 10; b is 1 to 10; x is 1 to 10; y is 1 to 10; z is 1 to 10 and B is K or R.

13. The method according to claim 1, wherein the labile peptide linker comprises of a polypeptide sequence of the format: --B-(G.sub.aS).sub.x--B'-- wherein a is 1 to 10; x is 1 to 10; B is K or R and B' is K or R.

14. The method according to claim 1, wherein the first immunoglobulin chain variable domain binds to a first target accessible via the intestinal tract.

15. The method according to claim 14, wherein the first target is a first deleterious agent originating from an intestinal tract resident pathogenic microbe.

16. The method according to claim 14, wherein the first target is selected from the group consisting of: an interleukin, an interleukin receptor, a transcription factor, a cytokine, a transmembrane protein, a surface glycoprotein, a soluble protein, an integrin, an adhesion molecule, a chemokine, a chemokine receptor, an inhibitory protein, a kinase, a G protein-coupled receptor or a toxin.

17. The method according to claim 1, wherein the first immunoglobulin chain variable domain is selected from the group consisting of: a VL, a V-NAR, an scFv, a Fab fragment, a F(ab')2 fragment, a VHH and a VH.

18. The method according to claim 17, wherein the second immunoglobulin chain variable domain is selected from the group consisting of: a VL, a V-NAR, an scFv, a Fab fragment, a F(ab')2 fragment, a VHH and a VH.

19. The method according to claim 1, wherein the labile peptide linker comprises a sequence selected from the group consisting of SEQ ID NOS: 2 to 5.

20. The method according to claim 19 wherein the labile peptide linker consists of a sequence selected from the group consisting of SEQ ID NOS: 2 to 5.
Description



CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation application of PCT/EP2016/057022 filed Mar. 31, 2016 which claims priority from EP 15162114.1 filed Mar. 31, 2015, EP 15162115.8 filed Mar. 31, 2015, EP 15162112.5 filed on Mar. 31, 2015 and EP 16152320.4 filed on Jan. 21, 2016, the contents of each of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to constructs suitable for oral administration comprising polypeptides connected by a labile peptide linker as well as to pharmaceutical compositions comprising such constructs. The present invention also relates to methods for preparing such constructs, methods which assay the lability of such constructs, methods which utilise such constructs, nucleic acids encoding such constructs, cDNA and vectors comprising nucleic acids encoding such constructs, host cells expressing or capable of expressing such constructs and to uses of such constructs or pharmaceutical compositions.

BACKGROUND OF THE INVENTION

[0003] Constructs comprising two or more polypeptides are a class of biomolecules with multi-functional properties. By genetically fusing two or more polypeptides together, the resultant construct may obtain many distinct functions derived from each component polypeptide. Such constructs have been utilised in biological research for many purposes such as protein purification and imaging. Such constructs have also become an important category of biopharmaceuticals. An effective construct requires a suitable linker. Direct fusion of polypeptides without a linker may lead to many undesirable outcomes including misfolding of the fused polypeptides, low yield in polypeptide production or impaired bioactivity. Therefore, the selection or rational design of a linker to join polypeptides is an important area in recombinant polypeptide technology.

[0004] Many constructs incorporate linkers which are relatively stable for the purposes of in vivo delivery or recombinant production. Stable linkers covalently join functional domains together to act as one molecule throughout the in vivo or recombinant production processes. A stable linkage between polypeptides may provide many advantages such as a prolonged plasma half-life and resistance to cleavage by host organism proteases. However, stable linkers also have several potential drawbacks including steric hindrance between polypeptides, decreased bioactivity, and altered biodistribution (Chen et al. 2013 Adv Drug Deliv Rev. 65(10):1357-1369). It would be advantageous therefore to release free polypeptides from a construct in vivo and thereby potentially reduce steric hindrance, improve bioactivity or achieve independent functions of individual polypeptides.

[0005] In the context of polypeptides having effects in the intestinal tract, such release would ideally take place in the intestinal tract after oral administration. It may be preferable that such release takes place only in one or a number of specific locations within the intestinal tract.

[0006] Constructs of the present invention may, in at least some embodiments, have one or more of the following advantages compared to substances of the prior art: [0007] (i) increased suitability for oral administration; [0008] (ii) increased suitability for local delivery to the intestinal tract following oral administration; [0009] (iii) ability to target (i.e. cleave the construct into component polypeptides) at one or more specific regions of the intestinal tract; [0010] (iv) increased convenience in incorporating two or more separable polypeptides in one recombinant product; [0011] (v) improved treatment and/or prevention of gastrointestinal tract infection or autoimmune and/or inflammatory diseases; [0012] (vi) increased suitability for expression, in a heterologous host such as bacteria (e.g. Escherichia coli) and/or mammalian cells and/or a yeast or mould (e.g. those belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia, such as Saccharomyces cerevisiae or Pichia pastoris); [0013] (vii) increased stability to protease degradation during production (for example resistance to yeast, mould or mammalian cell proteases); [0014] (viii) improved folding of polypeptides; [0015] (ix) improved yield during recombinant production; [0016] (x) improved bioactivity and/or biodistribution; [0017] (xi) suitability for, and improved properties for, use in a pharmaceutical; [0018] (xii) suitability for, and improved properties for, use in a functional food.

SUMMARY OF THE INVENTION

[0019] The present inventors have produced surprisingly advantageous constructs suitable for oral administration comprising a first polypeptide and a second polypeptide connected by a labile peptide linker. These constructs are particularly advantageous due to their convenience of production and their ability to release free component polypeptides within the intestinal tract. In one embodiment, the free component polypeptides may be released in one or more specific regions of the intestinal tract.

[0020] It may be expected that these constructs have particular utility in the prevention or treatment of diseases of the GIT such as autoimmune and/or inflammatory disease such as inflammatory bowel disease, or in the prevention or treatment of infection from an intestinal tract resident pathogenic microbe.

[0021] The present invention provides a construct suitable for oral administration comprising a first polypeptide and a second polypeptide connected by a labile peptide linker, wherein the labile peptide linker is labile to one or more proteases present in the intestinal tract and wherein the first and second polypeptides are substantially resistant to said one or more proteases. Also provided are related compositions, methods and nucleic acids relating to the inventive construct.

DESCRIPTION OF THE FIGURES

[0022] FIG. 1--Stained PAGE gel demonstrating non-lability of ID3A using the Trypsin Protease Assay

[0023] FIG. 2--Stained PAGE gel demonstrating lability of ID25A and ID26A using the Trypsin Protease Assay

[0024] FIG. 3--Stained PAGE gel demonstrating lability of ID27A and ID28A using the Trypsin Protease Assay

[0025] FIG. 4--Stained PAGE gel demonstrating lability of ID3A, ID25A, ID26A, ID27A and ID28A using the Faecal Protease Assay

[0026] FIG. 5--Stained PAGE gel demonstrating lability of ID55F, ID56F, ID57F, ID58F, ID59F and ID60F using the Faecal Protease Assay

[0027] FIG. 6--Stained PAGE gels demonstrating storage stability of ID3A and ID25A

[0028] FIG. 7--Stained PAGE gel demonstrating storage stability of ID26A, ID27A and ID28A

[0029] FIG. 8--Stained PAGE gel demonstrating storage stability of ID4A

DESCRIPTION OF THE SEQUENCES

[0030] SEQ ID NO: 1--Polypeptide sequence of linker used in constructs ID3A and ID55F [0031] SEQ ID NO: 2--Polypeptide sequence of linker used in constructs ID25A and ID57F [0032] SEQ ID NO: 3--Polypeptide sequence of linker used in constructs ID26A and ID58F [0033] SEQ ID NO: 4--Polypeptide sequence of linker used in constructs ID27A and ID59F [0034] SEQ ID NO: 5--Polypeptide sequence of linker used in constructs ID28A and ID60F [0035] SEQ ID NO: 6--Polypeptide sequence of linker used in construct ID56F [0036] SEQ ID NO: 7--Polypeptide sequence of ID3A construct [0037] SEQ ID NO: 8--Polypeptide sequence of ID25A construct [0038] SEQ ID NO: 9--Polypeptide sequence of ID26A construct [0039] SEQ ID NO: 10--Polypeptide sequence of ID27A construct [0040] SEQ ID NO: 11--Polypeptide sequence of ID28A construct [0041] SEQ ID NO: 12--Polypeptide sequence of ID55F construct [0042] SEQ ID NO: 13--Polypeptide sequence of ID57F construct [0043] SEQ ID NO: 14--Polypeptide sequence of ID58F construct [0044] SEQ ID NO: 15--Polypeptide sequence of ID59F construct [0045] SEQ ID NO: 16--Polypeptide sequence of ID60F construct [0046] SEQ ID NO: 17--Polypeptide sequence of ID56F construct [0047] SEQ ID NO: 18--Polypeptide sequence of ID1A ICVD [0048] SEQ ID NO: 19--Polypeptide sequence of ID5F ICVD [0049] SEQ ID NO: 20--Polynucleotide sequence encoding ID3A construct [0050] SEQ ID NO: 21--Polynucleotide sequence encoding ID25A construct [0051] SEQ ID NO: 22--Polynucleotide sequence encoding ID26A construct [0052] SEQ ID NO: 23--Polynucleotide sequence encoding ID27A construct [0053] SEQ ID NO: 24--Polynucleotide sequence encoding ID28A construct [0054] SEQ ID NO: 25--Polynucleotide sequence encoding ID55F construct [0055] SEQ ID NO: 26--Polynucleotide sequence encoding ID57F construct [0056] SEQ ID NO: 27--Polynucleotide sequence encoding ID58F construct [0057] SEQ ID NO: 28--Polynucleotide sequence encoding ID59F construct [0058] SEQ ID NO: 29--Polynucleotide sequence encoding ID60F construct [0059] SEQ ID NO: 30--Polynucleotide sequence encoding ID56F construct [0060] SEQ ID NO: 31--An exemplary polynucleotide sequence which would encode linker used in constructs ID3A and ID55F [0061] SEQ ID NO: 32--An exemplary polynucleotide sequence which would encode linker used in constructs ID25A and ID57F [0062] SEQ ID NO: 33--An exemplary polynucleotide sequence which would encode linker used in constructs ID26A and ID58F [0063] SEQ ID NO: 34--An exemplary polynucleotide sequence which would encode linker used in constructs ID27A and ID59F [0064] SEQ ID NO: 35--An exemplary polynucleotide sequence which would encode linker used in constructs ID28A and ID60F [0065] SEQ ID NO: 36--An exemplary polynucleotide sequence which would encode linker used in construct ID56F [0066] SEQ ID NO: 37--Proposed chymotrypsin-labile linker [0067] SEQ ID NO: 38--Polypeptide sequence of ID4A construct

DETAILED DESCRIPTION OF THE INVENTION

Labile Peptide Linkers

[0068] The construct of the invention comprises a labile peptide linker, which connects the first and second polypeptides. In one embodiment of the invention, the labile peptide linker can be engineered such that it resists cleavage by proteases to a desired extent and/or is only cleaved upon exposure to a specific area of the intestinal tract. For example, if a construct is recombinantly produced in a host such as yeast, trypsin-like proteases produced by the yeast may cleave the recombinant construct product. This may result in difficulties in purification and cause regulatory, clinical and commercial complications. Similarly, if for example the first polypeptide is a toxin, the second polypeptide of the construct may act to `quench` the effects of the toxin until it is released at a suitable, target location.

[0069] This can be achieved according to one embodiment of the invention by incorporating shielding residues into the labile peptide linker flanking the labile site(s). Shielding residues flank the labile site(s) of the labile peptide linker and reduce the lability thereof. Cleavage resistance can also be increased by positioning the labile site(s) closer to or at the periphery of the labile peptide linker. This concept is referred to as a "shielded labile site" and provides controlled lability.

[0070] In a further embodiment of the invention, the labile peptide linker can be engineered such that it is highly labile to cleavage by intestinal tract proteases, thereby quickly releasing the constituent first and second polypeptides of the construct after oral administration. This is achieved by incorporating one or more labile sites into the labile peptide linker such that the labile site is exposed for proteolysis, for example by positioning the labile site(s) substantially centrally in the labile peptide linker and/or by the labile site not being shielded substantially by flanking residues. This concept is referred to as a "non-shielded labile site".

[0071] Suitably the labile peptide linker has a length of at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 residues. Suitably the labile peptide linker has a length of no greater than 40, such as no greater than 35, such as no greater than 30, such as no greater than 25, such as no greater than 20, such as no greater than 15 residues.

[0072] Incorporation of a P residue into the labile peptide linker of the construct of the invention is expected to substantially prevent cleavage of the labile peptide linker. Suitably the labile peptide linker does not comprise any P residues.

Trypsin Labile Sites

Shielded Trypsin Labile Sites

[0073] Suitably the labile peptide linker of the construct of the invention comprises a cleavage site for trypsin or a trypsin-like protease. Suitably the labile peptide linker comprises at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 K residues. Suitably the labile peptide linker comprises at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 R residues. Preferably the cleavage site(s) is/are one or more K residue(s).

[0074] Shielding residues in the case of a trypsin or trypsin-like protease labile site may be D or E. Suitably the labile peptide linker comprises one or more shielding residues selected from the list consisting of D or E.

[0075] Suitably all K or R residues comprised within the labile peptide linker have at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5 shielding residues on their N-terminal side, wherein the shielding residues are selected from the list consisting of: D and E. Suitably all K or R residues comprised within the labile peptide linker have 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4, 4 to 5 shielding residues on their N-terminal side, wherein the shielding residues are selected from the list consisting of: D and E.

[0076] Suitably all K or R residues comprised within the labile peptide linker have and at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5 shielding residues on their C-terminal side, wherein the shielding residues are selected from the list consisting of: D and E. Suitably all K or R residues comprised within the labile peptide linker have 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4, 4 to 5 shielding residues on their C-terminal side, wherein the shielding residues are selected from the list consisting of: D and E.

[0077] Suitably all K and R residues have at least one shielding residue adjacent to them, suitably followed by one or more further contiguous shielding residues. Suitably the shielding residues occur on one or both sides of one or more of the K or R residues.

[0078] Suitably the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:

[-(G.sub.aS).sub.x--U.sub.c--B--U'.sub.d-(G.sub.bS).sub.y--].sub.z [0079] wherein [0080] a is 1 to 10; [0081] b is 1 to 10; [0082] U is D or E; [0083] U' is D or E; [0084] c is 0 to 7; [0085] d is 0 to 7; [0086] x is 1 to 10; [0087] y is 1 to 10 [0088] z is 1 to 10 and [0089] B is K or R.

[0090] Suitably a is 2 to 5, more suitably a is 4. Suitably b is 2 to 5, more suitably b is 4. Suitably x is 1 to 5, more suitably x is 1. Suitably y is 1 to 5, more suitably y is 1. Suitably z is 1 to 3, more suitably z is 1. Suitably, B is K. Suitably, U if present, is D. Suitably, U' if present, is D. In one embodiment c is 1 and d is 1. In another embodiment c is 0 and d is 0. In a further embodiment c is 4 and d is 0. Suitably both U and U' are each individually D and c and d are both 1.

[0091] Suitably the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:

--B-(G.sub.aS).sub.x--B'-- [0092] wherein [0093] a is 1 to 10; [0094] x is 1 to 10; [0095] B is K or R and [0096] B' is K or R.

[0097] In one embodiment, a is 2 to 5, more suitably a is 4. In a further embodiment x is 1 to 5. More suitably, x is 2. Suitably, B is K. Suitably, B' is K.

[0098] Suitably the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:

--B-(G.sub.aS).sub.x--B'-(G.sub.bS).sub.y--B''-- [0099] wherein [0100] a is 1 to 10; [0101] b is 1 to 10; [0102] x is 1 to 10; [0103] y is 1 to 10; [0104] B is K or R [0105] B' is K or R and [0106] B'' is K or R.

[0107] In one embodiment, a is 2 to 5, more suitably a is 4. In one embodiment, b is 2 to 5, more suitably b is 4. In a further embodiment x is 1 to 5. More suitably, x is 2. In a further embodiment y is 1 to 5. More suitably, y is 2. Suitably, B is K. Suitably, B' is K. Suitably, B'' is K.

Non-Shielded Trypsin Labile Sites

[0108] Suitably the labile peptide linker of the construct of the invention comprises a cleavage site for trypsin or a trypsin-like protease. Suitably the labile peptide linker comprises at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 K residues. Suitably the labile peptide linker comprises at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 R residues. Preferably the cleavage site(s) is/are one or more K residue(s).

[0109] Suitably all K or R residues comprised within the labile peptide linker have at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5 non-shielding residues on their N-terminal side wherein the non-shielding residues are selected from the list consisting of: C, A, S, N, G, L, I, V, T, M, F, Y, H, W and Q; more suitably A, G, L, I, V, M, S and T; more suitably A, G, L, I, V and S; more suitably G and S. Suitably all K or R residues comprised within the labile peptide linker have 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4, 4 to 5 non-shielding residues on their N-terminal side, wherein the shielding residues are selected from the list consisting of: C, A, S, N, G, L, I, V, T, M, F, Y, H, W and Q; more suitably A, G, L, I, V, M, S and T; more suitably A, G, L, I, V and S; more suitably G and S.

[0110] Suitably all K or R residues comprised within the labile peptide linker have at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5 non-shielding residues on their C-terminal side, wherein the non-shielding residues are selected from the list consisting of: C, A, S, N, G, L, I, V, T, M, F, Y, H, W and Q; more suitably A, G, L, I, V, M, S and T; more suitably A, G, L, I, V and S; more suitably G and S. Suitably all K or R residues comprised within the labile peptide linker have 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4, 4 to 5 non-shielding residues on their C-terminal side, wherein the shielding residues are selected from the list consisting of: C, A, S, N, G, L, I, V, T, M, F, Y, H, W and Q; more suitably A, G, L, I, V, M, S and T; more suitably A, G, L, I, V and S; more suitably G and S.

[0111] Suitably all K and R residues have at least one non-shielding residue adjacent to them, suitably followed by one or more further contiguous non-shielding residues. Suitably the non-shielding residues occur on one or both sides of one or more of the K or R residues.

[0112] Suitably the labile peptide linker does not comprise any D or E residues. Suitably the labile peptide linker consists of residues selected from the list consisting of C, A, S, N, G, L, I, V, T, M, F, Y, H, K, R, W and Q; more suitably A, G, L, I, V, M, S, T, K and R residues; more suitably S, G, K and R residues.

[0113] Suitably the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:

[-(G.sub.aS).sub.x--B-(G.sub.bS).sub.y--].sub.z [0114] wherein [0115] a is 1 to 10; [0116] b is 1 to 10; [0117] x is 1 to 10; [0118] y is 1 to 10 [0119] z is 1 to 10 and [0120] B is K or R.

[0121] Suitably a is 2 to 5, more suitably a is 4. Suitably b is 2 to 5, more suitably b is 4. Suitably x is 1 to 5, more suitably x is 1. Suitably y is 1 to 5, more suitably y is 1. Suitably z is 1 to 3, more suitably z is 1. Suitably B is K.

[0122] Suitably the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:

[-(G.sub.4S).sub.x--B-(G.sub.4S).sub.y--].sub.z [0123] wherein [0124] x is 1 to 10; [0125] y is 1 to 10 [0126] z is 1 to 10 and [0127] B is K or R.

[0128] Suitably x is 1 to 5, more suitably x is 1. Suitably y is 1 to 5, more suitably y is 1. Suitably z is 1 to 3, more suitably, z is 1. Suitably B is K.

Chymotrypsin Labile Sites

Non-Shielded Chymotrypsin Labile Sites

[0129] Alternatively, or in addition to trypsin labile sites, the labile peptide linker of the construct of the invention comprises a cleavage site for chymotrypsin or a chymotrypsin-like protease. Suitably the labile peptide linker comprises at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 residues selected from the list consisting of W, F, Y, L and M; more suitably W, F and Y. Suitably the labile peptide linker consists of residues selected from the list consisting of S, G, W, F, Y, L and M; such as S, G, W, F and Y.

[0130] Suitably the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:

[-(G.sub.aS).sub.x-J-(G.sub.bS).sub.y--].sub.z [0131] wherein [0132] a is 1 to 10; [0133] b is 1 to 10; [0134] x is 1 to 10; [0135] y is 1 to 10 [0136] z is 1 to 10 and [0137] J is W, F, Y, L or M; such as W, F or Y.

[0138] In one embodiment a is 2 to 5, in a further embodiment, b is 2 to 5, in a further embodiment x is 1 to 5, in a further embodiment, y is 1 to 5, in a further embodiment z is 1 to 3. Suitably x is 1, y is 1 and z is 1.

[0139] Suitably the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:

[-(G.sub.4S).sub.x-J-(G.sub.4S).sub.y--].sub.z [0140] wherein [0141] x is 1 to 10; [0142] y is 1 to 10 [0143] z is 1 to 10 and [0144] J is W, F, Y, L or M; such as W, F or Y.

[0145] In one embodiment, x is 1 to 5, in a further embodiment, y is 1 to 5, in a further embodiment z is 1 to 3. Suitably x is 1, y is 1 and z is 1.

MMP Labile Sites

[0146] In one embodiment, the labile peptide linker of the construct of the invention comprises a cleavage site for MMP3, MMP10 or MMP12.

Lability

[0147] The lability of a construct, and therefore the utility of a construct as a controlled-lability or highly labile construct can be assayed using the Faecal Protease Assay, the Trypsin Protease Assay and/or the Chymotrypsin Protease Assay defined in Examples 1 to 3.

[0148] Suitably at least 20%, such as at least 40%, such as at least 60%, such as at least 80%, such as at least 90%, such as at least 100% by mass of the construct remains uncleaved after at least 10, such as at least 20, such as at least 30, such as at least 40, such as at least 50, such as at least 60, such as at least 70, such as at least 80, such as at least 90, such as at least 100, such as at least 110, such as at least 120, such as at least 130, such as at least 140, such as at least 150, such as at least 160 minutes after mixing in the Faecal Protease Assay and/or Trypsin Protease Assay and/or Chymotrypsin Protease Assay.

[0149] Alternatively, at least 60%, such as at least 80%, such as at least 90%, such as at least 100% of the construct is cleaved after no more than 5, such as no more than 4, such as no more than 3, such as no more than 2, such as no more than 1 minute, after mixing in the Faecal Protease Assay and/or Trypsin Protease Assay and/or Chymotrypsin Protease Assay.

[0150] In one aspect of the invention there is provided a method of assaying the lability of a construct of the invention, comprising the steps of: (a) incubating the construct in a solution comprising trypsin, a solution comprising chymotrypsin, faecal supernatant, small intestinal fluid or a solution comprising enteropeptidase (such as by performing the Trypsin Protease Assay, the Chymotrypsin Protease Assay or the Faecal Protease Assay) then (b) ascertaining the proportion of cleaved construct after one or more periods of incubation.

[0151] In a further aspect of the invention there is provided a method of delivering a monomeric antibody or a monomeric antigen binding fragment thereof to a targeted region of the intestinal tract, comprising the steps of: (a) performing the method of assaying construct lability described above, (b) selecting a construct with an appropriate level of lability for the targeted region of the intestinal tract, (c) producing the selected construct with an enterically coated packaging then (d) administering the packaged selected construct to a subject.

[0152] In a further aspect of the invention there is provided a method of preparing a product comprising a construct of the invention which has been selected, the method comprising adding the selected construct into the product, wherein the selected construct is selected and produced by a method comprising the steps of: (a) performing the method of assaying construct lability described above then (b) selecting a construct with an appropriate level of lability for the targeted region of the intestinal tract.

Stability

[0153] Various organisms may be used to express recombinant polypeptides. Commonly used expression organisms include yeast, mould and mammalian cells. However, many of these expression organisms also produce proteases, such as trypsin-like proteases, which may cleave the expressed recombinant polypeptide. If the expressed polypeptide incorporates a peptide linker which is labile to one or more proteases present in the intestinal tract, then this peptide linker may undesirably also be labile to proteases produced by the expression organism, thus preventing effective expression of intact polypeptide.

[0154] It is advantageous for the labile peptide linker to be substantially non-labile to enzymes produced by the recombinant host used to produce the construct. Suitably the labile peptide linker is substantially resistant to proteases produced by a recombinant host such as bacteria such as E. coli or such as a yeast or mould belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia; such as Saccharomyces cerevisiae or Pichia pastoris. Suitably the recombinant host is a yeast. Suitably the recombinant host is a mould. Suitably the yeast belongs to the genera Saccharomyces, Kluyveromyces, Hansenula or Pichia. Further examples of yeasts are those belonging to the genera Candida and Torulopsis. Suitably the mould belongs to the genus Aspergillus. Further examples of moulds are those belonging to the genera Acremonium, Alternaria, Chrysosporium, Cladosporium, Dictyostelium, Fusarium, Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma and Trichophyton. Suitably the labile peptide linker is substantially resistant to proteases produced by a recombinant host for at least 2, such as at least 4, such as at least 9, such as at least 14, such as at least 60 days' storage at 4 degrees C.

Yeast-Produced Constructs for Oral Administration

[0155] In one aspect of the invention there is provided a construct for use in the treatment by oral administration of a disease of the intestinal tract with a first immunoglobulin chain variable domain and a second immunoglobulin chain variable domain, wherein the construct comprises the first immunoglobulin chain variable domain and the second immunoglobulin chain variable domain connected by a labile peptide linker, wherein: [0156] (i) the labile peptide linker is labile to one or more proteases present in the intestinal tract, [0157] (ii) the labile peptide linker is stable to yeast proteases and [0158] (iii) the first and second immunoglobulin chain variable domains are substantially resistant to said one or more proteases and wherein the construct is produced in yeast.

[0159] In a further aspect of the invention there is provided a method of treating by oral administration a disease of the intestinal tract with a first immunoglobulin chain variable domain and a second immunoglobulin chain variable domain from a construct, wherein the construct comprises the first immunoglobulin chain variable domain and the second immunoglobulin chain variable domain connected by a labile peptide linker, wherein: [0160] (i) the labile peptide linker is labile to one or more proteases present in the intestinal tract, [0161] (ii) the labile peptide linker is stable to yeast proteases and [0162] (iii) the first and second immunoglobulin chain variable domains are substantially resistant to said one or more proteases; wherein the construct is produced in yeast.

[0163] In a further aspect of the invention there is provided a method of delivering a first immunoglobulin chain variable domain and a second immunoglobulin chain variable domain to the intestinal tract comprising producing in yeast and then orally administering a construct comprising the first immunoglobulin chain variable domain and the second immunoglobulin chain variable domain connected by a labile peptide linker, wherein: [0164] (i) the labile peptide linker is labile to one or more proteases present in the intestinal tract, [0165] (ii) the labile peptide linker is stable to yeast proteases and [0166] (iii) the first and second immunoglobulin chain variable domains are substantially resistant to said one or more proteases.

[0167] In a further aspect of the invention there is provided a method of making a construct comprising a first immunoglobulin chain variable domain and a second immunoglobulin chain variable domain connected by a labile peptide linker, wherein: [0168] (i) the labile peptide linker is labile to one or more proteases present in the intestinal tract, [0169] (ii) the labile peptide linker is stable to yeast proteases and [0170] (iii) the first and second immunoglobulin chain variable domains are substantially resistant to said one or more proteases; comprising the step of producing the construct in yeast.

[0171] Suitably the step of producing the construct in yeast is performed by providing a host yeast cell which is capable of expressing the construct of the invention, transformed with a vector, wherein the vector comprises a polynucleotide encoding the construct of the invention and wherein the host yeast cell is exposed to conditions suitable for expression of the construct of the invention.

[0172] Suitably the first immunoglobulin chain variable domain and the second immunoglobulin chain variable domain are substantially resistant to yeast proteases.

[0173] Suitably the methods outlined above further comprise the step of purifying the construct. Suitably the labile peptide linker is cleaved in the intestinal tract by the one or more proteases present in the intestinal tract. In a further aspect of the invention, there is provided a construct obtained by any of the above methods.

[0174] The constructs for use and methods described above relate to the production of a construct in yeast. However, these constructs for use and methods are also equally applicable to production of a construct in any expression organism which produces proteases which may cleave a peptide linker, such as mammalian cells or moulds (such as moulds from any one or more of the genera Acremonium, Alternaria, Aspergillus, Chrysosporium, Cladosporium, Dictyostelium, Fusarium, Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma and Trichophyton).

[0175] Particular characteristics of these embodiments of the invention are further discussed as follows.

1. A Labile Peptide Linker which is Labile to One or More Proteases Present in the Intestinal Tract

[0176] Examples 5 and 6 below provide details on specific constructs of the invention which comprise labile peptide linkers which are labile to one or more proteases present in the intestinal tract.

[0177] Suitably the labile peptide linker is labile to one or more proteases present in the intestinal tract such that greater than 50%, such as greater than 60%, such as greater than 70%, such as greater than 80%, such as greater than 85%, such as greater than 90% by mass of the construct is cleaved into first and second immunoglobulin chain variable domains after 160 minutes, more suitably after 105 minutes, more suitably after 60 minutes, more suitably after 25 minutes, more suitably after 10 minutes after mixing in the Trypsin Protease Assay.

2. A Labile Peptide Linker which is Stable to Yeast Proteases

[0178] Example 7 below provides details on specific constructs of the invention which comprise labile peptide linkers which are stable to yeast proteases. Example 9 below provides details on a specific construct (ID4A) which is not stable to yeast proteases.

[0179] The stability of a linker to yeast proteases may be assessed using the Yeast Expression Protocol outlined as follows.

The Yeast Expression Protocol:

[0180] The following protocol outlines a method for the cloning of polypeptides (such as constructs comprising ICVDs or monomeric ICVDs) into the chromosome of Saccharomyces cerevisiae such that induction in a suitable growth medium results in polypeptide expression and secretion into the extracellular supernatant.

[0181] An S. cerevisiae strain of the following description is used for this process: The production strain used for expression and manufacture of polypeptides is a derivative of the CEN.PK series from the EUROSCARF collection (EUROpean Saccharomyces Cerevisiae ARchive for Functional analysis). The genotype of the CEN.PK strain is: MATa/MAT.alpha. ura3-52/ura3-52; trp1-289/trp1-289; leu2-3,112/leu2-3,112; his3.DELTA. 1/his3.DELTA. 1; MAL2-8C/MAL2-8C; SUC2/SUC2. This strain is then further modified to inhibit the ability to grow on galactose by deletion of the galactokinase gene (gal1::URA3). This is the final strain used for transformation by the polypeptide expression constructs.

[0182] Monomeric or multimeric DNA constructs, in which polypeptides (such as constructs comprising ICVDs or monomeric ICVDs) joined by protein linkers, are cloned into a suitable multi-copy chromosomal integration vector (Lopes et al. 1991. Gene. 105, 83-90), generating an integration cassette. The integration cassette may include an inducible promoter (for example pGal7, Nogi & Fukasawa (1983). Nucleic Acids Res. 11(24):8555-68), the polypeptide encoding region, a signal sequence that encodes secretion into the extracellular supernatant in yeast (Hashimoto et al. 1998. Protein Engineering.11 (75-77)) fused immediately upstream of the first amino acid in the polypeptide coding region, an auxotrophic selection marker and DNA sequences that contribute to recombination into the chromosome. Transformation of competent yeast cells with linear DNA encoding the integration cassette and subsequent selection on a suitable auxotrophic medium (for example omitting leucine where a leucine biosynthesis gene is the selection marker) results in integration and amplification at the rDNA locus, such that 100-200 copies of the expression construct may be present in the cell. Following the removal of the selective pressure the expression construct remains stably integrated into the chromosome. Alternatively, polypeptide production may be achieved from a multi-copy episomal vector based on the yeast 2 .mu.M plasmid with a similar expression cassette, without the need for chromosomal integration.

[0183] To induce polypeptide expression, a colony of the resulting yeast strain is inoculated into 5 mL of yeast soytone broth supplemented with 2% glucose and grown overnight at 30.degree. C. (150-200 rpm). The following day 50 mL of yeast soytone broth containing 2% glucose and 0.5% galactose (for induction) in a 500 mL Erlenmeyer flask is inoculated with the entire 5 mL overnight culture. The resulting induction culture is incubated at 30.degree. C., 200 rpm for 3 days. The culture is then spun down at 4200 rpm for 20 min in a swing rotor centrifuge to remove yeast cells. The supernatant is then filtered through 0.45 .mu.m and 0.2 .mu.m filters in series.

[0184] After carrying out the Yeast Expression Protocol, the polypeptide may then optionally undergo a storage period at 4.degree. C. in the yeast supernatant under sterile conditions.

[0185] Suitably a construct of the invention is stable to yeast proteases such that no more than 10%, more suitably no more than 5%, more suitably no more than 1% by mass of the construct is cleaved into first and second immunoglobulin chain variable domains after producing the construct using the Yeast Expression Protocol (i.e. with no subsequent storage period) and then optionally storing for 2 days, more suitably 4 days or more suitably 9 days. "0 days storage" as used herein refers to no subsequent storage period.

[0186] Yeast-produced polypeptides may be distinct from those produced using alternative expression organisms such as bacteria, in that yeast-produced polypeptides may comprise post translational modifications such as glycosylation.

3. A First and Second Immunoglobulin Chain Variable Domain which are Substantially Resistant to One or More Proteases Present in the Intestinal Tract

[0187] Examples 5 and 6 below provide details on specific constructs of the invention which comprise first and second immunoglobulin chain variable domains, wherein the first and second immunoglobulin chain variable domains are substantially resistant to one or more proteases present in the intestinal tract.

[0188] Suitably the first immunoglobulin chain variable domain and the second immunoglobulin chain variable domain are substantially resistant to one or more proteases present in the intestinal tract such that at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as about 100% by mass of the first immunoglobulin chain variable domain and at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as about 100% by mass of the second immunoglobulin chain variable domain remain uncleaved after 10 minutes, more suitably after 25 minutes, more suitably after 60 minutes, more suitably after 105 minutes, more suitably after 160 minutes after mixing in the Trypsin Protease Assay.

4. A First and Second Immunoglobulin Chain Variable Domain which are Substantially Resistant to Yeast Proteases

[0189] Example 7 below provides details on specific constructs of the invention which comprise immunoglobulin chain variable domains, wherein the immunoglobulin chain variable domains are stable to yeast proteases.

[0190] Suitably the first immunoglobulin chain variable domain and the second immunoglobulin chain variable domain are substantially resistant to yeast proteases such that no more than 10%, more suitably no more than 5%, more suitably no more than 1% by mass of the first or second immunoglobulin chain variable domain are cleaved after producing the first or second immunoglobulin chain variable domain using the Yeast Expression Protocol and then optionally storing for 2 days, more suitably 4 days or more suitably 9 days.

[0191] Assessment of Stability and Lability Under Points 1-4 Above

[0192] Suitably the cleaved mass of the construct and/or the uncleaved mass of the first and second immunoglobulin chain variable domains and/or the cleaved mass of the first and second immunoglobulin chain variable domains are assessed by gel electrophoresis followed by visual inspection of the gel. More suitably, assessment is by quantitative gel electrophoresis. Alternatively, assessment is by gel electrophoresis followed by mass spectrometry.

[0193] Bands on a gel corresponding to cleaved construct and uncleaved immunoglobulin chain variable domain may be identified as those bands having approximately half the mass of the construct (i.e. the mass of one constituent immunoglobulin chain variable domain), by comparison with molecular weight marker. Bands on a gel corresponding to cleaved immunoglobulin chain variable domains are identifiable by having a molecular weight lower than that of the whole constituent immunoglobulin chain variable domain, by comparison to molecular weight marker bands.

[0194] In one embodiment, the labile peptide linker of the construct of the invention does not comprise a polypeptide disclosed in WO2009/021754. More specifically, in one embodiment the labile peptide linker of the construct of the invention does not comprise the sequence GGGGSDDDDKGGGGS (SEQ ID NO: 4).

Polypeptides, Antigen-Binding Polypeptides, Antibodies and Antibody Fragments Including Immunoglobulin Chain Variable Domains (ICVD) Such as the VH and VHH

[0195] Polypeptides are organic polymers consisting of a number of amino acid residues bonded together in a chain. As used herein, `polypeptide` is used interchangeably with `protein` and `peptide`. Polypeptides are said to be antigen-binding when they contain one or more stretches of amino acid residues which form an antigen-binding site, capable of binding to an epitope on a target antigen with an affinity (suitably expressed as a Kd value, a Ka value, a k.sub.on-rate and/or a k.sub.off-rate, as further described herein). Antigen-binding polypeptides include polypeptides such as antibodies, antibody fragments and antigen-binding fragments. A polypeptide may comprise a region which is capable of binding a target with high affinity (suitably expressed as a Kd value, a Ka value, a k.sub.on-rate and/or a k.sub.off-rate, as further described herein). Such polypeptides include DARPins (Binz et al. Journal of Molecular Biology 332(2):489-503), Affimers.TM., Fynomers.TM., Centyrins, Nanofitins.RTM. and cyclic peptides.

[0196] A conventional antibody or immunoglobulin (Ig) is a protein comprising four polypeptide chains: two heavy (H) chains and two light (L) chains. Each chain is divided into a constant region and a variable domain. The heavy chain variable domains are abbreviated herein as VHC, and the light (L) chain variable domains are abbreviated herein as VLC. These domains, domains related thereto and domains derived therefrom, are referred to herein as immunoglobulin chain variable domains. The VHC and VLC domains can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDRs"), interspersed with regions that are more conserved, termed "framework regions" ("FRs"). The framework and complementarity determining regions have been precisely defined (Kabat et al 1991 Sequences of Proteins of Immunological Interest, Fifth Edition U.S. Department of Health and Human Services, NIH Publication Number 91-3242, herein incorporated by reference in its entirety). In a conventional antibody, each VHC and VLC is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The conventional antibody tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains is formed with the heavy and the light immunoglobulin chains inter-connected by e.g. disulfide bonds, and the heavy chains similarity connected. The heavy chain constant region includes three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable domain of the heavy chains and the variable domain of the light chains are binding domains that interact with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (C1q) of the classical complement system. The term antibody includes immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be kappa or lambda types. The overall structure of immunoglobulin-gamma (IgG) antibodies assembled from two identical heavy (H)-chain and two identical light (L)-chain polypeptides is well established and highly conserved in mammals (Padlan 1994 Mol Immunol 31:169-217).

[0197] An exception to conventional antibody structure is found in sera of Camelidae. In addition to conventional antibodies, these sera possess special IgG antibodies. These IgG antibodies, known as heavy-chain antibodies (HCAbs), are devoid of the L chain polypeptide and lack the first constant domain (CH1). At its N-terminal region, the H chain of the homodimeric protein contains a dedicated immunoglobulin chain variable domain, referred to as the VHH, which serves to associate with its cognate antigen (Muyldermans 2013 Annu Rev Biochem 82:775-797, Hamers-Casterman et al 1993 Nature 363(6428):446-448, Muyldermans et al 1994 Protein Eng 7(9):1129-1135, herein incorporated by reference in their entirety).

[0198] An antigen-binding fragment (or "`antibody fragment" or "immunoglobulin fragment") as used herein refers to a portion of an antibody that specifically binds to a target (e.g. a molecule in which one or more immunoglobulin chains is not full length, but which specifically binds to a target). Examples of binding fragments encompassed within the term antigen-binding fragment include: [0199] (i) a Fab fragment (a monovalent fragment consisting of the VLC, VHC, CL and CH1 domains); [0200] (ii) a F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region); [0201] (iii) a Fd fragment (consisting of the VHC and CH1 domains); [0202] (iv) a Fv fragment (consisting of the VLC and VHC domains of a single arm of an antibody); [0203] (v) an scFv fragment (consisting of VLC and VHC domains joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLC and VHC regions pair to form monovalent molecules); [0204] (vi) a VH (an immunoglobulin chain variable domain consisting of a VHC domain (Ward et al Nature 1989 341:544-546); [0205] (vii) a VL (an immunoglobulin chain variable domain consisting of a VLC domain); [0206] (viii) a V-NAR (an immunoglobulin chain variable domain consisting of a VHC domain from chondrichthyes IgNAR (Roux et al 1998 Proc Natl Acad Sci USA 95:11804-11809 and Griffiths et al 2013 Antibodies 2:66-81, herein incorporated by reference in their entirety) [0207] (ix) a VHH.

[0208] The total number of amino acid residues in a VHH or VH may be in the region of 110-130, is suitably 112-120, and is most suitably 115.

[0209] Constructs of the invention comprising polypeptides and labile peptide linkers may for example be obtained by preparing a nucleic acid encoding two or more polypeptides and a labile peptide linker using techniques for nucleic acid synthesis, followed by expression of the nucleic acid thus obtained (as detailed further herein). According to a specific embodiment, a construct according to the invention does not have an amino acid sequence which is exactly the same as (i.e. shares 100% sequence identity with) the amino acid sequence of a naturally occurring polypeptide.

[0210] Suitably the first and/or second polypeptide of the construct of the invention is an antigen-binding polypeptide, more suitably the first and/or second antigen-binding polypeptide is an immunoglobulin chain variable domain, an antibody or an antigen-binding fragment thereof. More suitably the antigen-binding fragment is selected from the group consisting of: VLs, V-NARs, scFvs, Fab fragments, F(ab')2 fragments or immunoglobulin chain variable domains such as VHHs and VHs.

Specificity, Affinity and Avidity

[0211] Specificity refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding polypeptide can bind. The specificity of an antigen-binding polypeptide is the ability of the antigen-binding polypeptide to recognise a particular antigen as a unique molecular entity and distinguish it from another.

[0212] Affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding polypeptide (Kd), is a measure of the binding strength between an antigenic determinant and an antigen-binding site on an antigen-binding polypeptide: the lesser the value of the Kd, the stronger the binding strength between an antigenic determinant and the antigen-binding polypeptide (alternatively, the affinity can also be expressed as the affinity constant (Ka), which is 1/Kd). Affinity can be determined by known methods, depending on the specific antigen of interest.

[0213] Avidity is the measure of the strength of binding between an antigen-binding polypeptide and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen-binding site on the antigen-binding polypeptide and the number of pertinent binding sites present on the antigen-binding polypeptide.

[0214] Suitably, antigen-binding polypeptides bind with a dissociation constant (Kd) of 10.sup.-6 to 10.sup.-12 M, more suitably 10.sup.-7 to 10.sup.-12 M, more suitably 10.sup.-8 to 10.sup.-12 M and more suitably 10.sup.-9 to 10.sup.-12 M.

[0215] Any Kd value less than 10.sup.-6 is considered to indicate binding. Specific binding of an antigen-binding polypeptide to an antigen or antigenic determinant can be determined in any suitable known manner, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known in the art.

Polypeptide and Polynucleotide Sequences

[0216] The construct of the invention, the labile peptide linker and the first and second polypeptides comprised within the construct all comprise amino acid residues.

[0217] Suitably the first and second polypeptides each comprise or more suitably consists of a sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 19

[0218] Suitably the construct comprises or more suitably consists of a sequence selected from the group consisting of SEQ ID NOs: 7 to 17. Suitably the construct comprises or more suitably consists of a sequence selected from the group consisting of SEQ ID NOs: 8 to 11 and 13 to 16.

[0219] Suitably the labile peptide linker comprises or more suitably consists of a sequence selected from the group consisting of SEQ ID NOs: 1 to 6. Suitably the labile peptide linker comprises or more suitably consists of a sequence selected from the group consisting of SEQ ID NOs: 2 to 5.

[0220] An amino acid residue in regions of the construct other than the labile peptide linker can be replaced with another amino acid residue of similar chemical structure and which is expected to have little influence on the function, activity or other biological properties of the polypeptide. Such a substitution is a "conservative" substitution Such conservative substitutions suitably are substitutions in which one amino acid within the following groups is substituted by another amino acid residue from within the same group:

TABLE-US-00001 Group Amino acid residue Non-polar aliphatic Glycine Alanine Valine Leucine Isoleucine Aromatic Phenylalanine Tyrosine Tryptophan Polar uncharged Serine Threonine Asparagine Glutamine Negatively charged Aspartate Glutamate Positively charged Lysine Arginine

[0221] The construct, excluding the labile peptide linker, may suitably include one or more conservative substitutions.

[0222] In one aspect of the invention there is provided a nucleic acid encoding the construct of the invention.

[0223] For the avoidance of doubt, the single-letter amino acid code is as follows: [0224] G--Glycine (Gly), P--Proline (Pro), A--Alanine (Ala), V--Valine (Val), L--Leucine (Leu), I--Isoleucine (Ile), M--Methionine (Met), C--Cysteine (Cys), F--Phenylalanine (Phe), Y--Tyrosine (Tyr), W--Tryptophan (Trp), H--Histidine (His), K--Lysine (Lys), R--Arginine (Arg), Q--Glutamine (Gin), N--Asparagine (Asn), E--Glutamic Acid (Glu), D--Aspartic Acid (Asp), S--Serine (Ser), T--Threonine (Thr).

Multimers

[0225] A construct according to the invention comprises a first polypeptide and a second polypeptide. The inventive construct is therefore multimeric and may suitably be multivalent. Such a construct may comprise a first polypeptide and a second polypeptide which are identical. A construct consisting of two identical polypeptides is a "homobihead". In one aspect of the invention there is provided a construct comprising two identical polypeptides. Alternatively, a construct may consist of a first polypeptide and a second polypeptide which are different from one another (a "heterobihead").

[0226] Constructs can be multivalent and/or multispecific. A multivalent construct (such as a bivalent construct) comprises two or more binding polypeptides and therefore provides two or more sites at which attachment to antigens can occur suitably before or after cleavage of the labile peptide linker. An example of a multivalent construct could be a homobihead or a heterobihead. A multispecific construct such as a bispecific construct comprises two different binding polypeptides which present two sites at which either (a) attachment to two different antigens can occur or (b) attachment to two different epitopes on the same antigen can occur, suitably before or after cleavage of the labile peptide linker. An example of a multispecific construct could be a heterobihead. A multispecific construct is multivalent.

[0227] A construct of the invention may comprise an additional third polypeptide (connected to the first polypeptide by a peptide linker) and may also comprise or consist of an additional fourth polypeptide (connected to the second polypeptide by a peptide linker), wherein the third and fourth polypeptides are substantially resistant to the one or more proteases present in the intestinal tract and are as defined herein in respect of the first and second polypeptides. A construct of the invention consisting of four polypeptides is known as a `quadrahead`. Suitably, the peptide linkers are substantially resistant to the one or more proteases present in the intestinal tract or alternatively the peptide linkers are labile peptide linkers as defined herein.

[0228] Suitably the first, second, third and/or fourth polypeptide has a molecular weight of no greater than 300 kDa, such as 250 kDa, such as 200 kDa, such as 180 kDa, such as 160 kDa, such as 140 kDa, such as 120 kDa, such as 100 kDa, such as 80 kDa, such as 60 kDa.

The Gastrointestinal Tract and Digestive Enzymes

[0229] The gastrointestinal tract (GIT) is an organ system responsible for consuming and digesting foodstuffs, absorbing nutrients, and expelling waste. In humans and other mammals, the GIT consists of the oesophagus, stomach, small intestine (duodenum, jejunum and ileum) and large intestine (cecum, colon, rectum and anal canal). Various pathogens may colonise and various diseases may manifest in different areas of the GIT. The intestinal tract (as opposed to the gastrointestinal tract) consists of the small and large intestine.

[0230] The different parts of the gastrointestinal tract each contain a complex mixture of digestive enzymes. These digestive enzymes include proteases, lipases, amylases and nucleases. Proteases include serine proteases, threonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases and metalloproteases. Proteases are involved in digesting polypeptide chains into shorter fragments by splitting the peptide bonds that link amino acid residues (proteolysis). Some detach the terminal amino acids from the protein chain (exopeptidases), others attack internal peptide bonds of a protein (endopeptidases).

[0231] Proteolysis can be highly promiscuous such that a wide range of protein substrates are hydrolysed. This is the case for proteases which cleave the wide array of ingested polypeptides in the intestinal tract into smaller polypeptide fragments.

[0232] Many proteases typically bind to a single amino acid (a labile site) on the substrate and so only have specificity for that residue. The proteases present in the intestinal tract include trypsin, trypsin-like proteases, chymotrypsin, chymotrypsin-like proteases, carboxypeptidase, elastase, aminopeptidase, carboxypeptidase and enteropeptidase. Trypsin-like proteases cleave peptide bonds following lysine or arginine residues. Chymotrypsin-like proteases cleave peptide bonds following hydrophobic residues, such as tyrosine, phenylalanine, tryptophan, leucine and methionine. Particularly tyrosine, phenylalanine and tryptophan.

[0233] Suitably the labile peptide linker is labile to one or more proteases present in the intestinal tract and wherein the first and second polypeptides are substantially resistant to said one or more proteases, wherein said one or more proteases are present in the small or large intestine, more suitably the jejunum, the ileum and/or the cecum. Suitably the one or more proteases are serine proteases. Suitably the one or more proteases are selected from the group consisting of enteropeptidase, trypsin, trypsin-like proteases, chymotrypsin and chymotrypsin-like proteases.

[0234] Suitably the first and second polypeptides of the construct of the invention are substantially resistant to all proteases present in the intestinal tract. Such proteases include proteases sourced from gastrointestinal tract commensal microflora or pathogenic bacteria, for example wherein the proteases are cell membrane-attached proteases, secreted proteases and proteases released on cell lysis. Suitably the intestinal tract is a mammalian intestinal tract, such as a human, simian, murine, bovine, ovine or porcine intestinal tract.

Diseases of the GIT

[0235] Diseases of the GIT refer to diseases involving the gastrointestinal tract, namely the oesophagus, stomach, small intestine (duodenum, jejunum and ileum) and large intestine (cecum, colon, rectum and anal canal). The construct of the invention may be used in the treatment or prevention of such diseases. Exemplary diseases of the GIT are described below.

Autoimmune Diseases and/or Inflammatory Diseases of the GIT

[0236] Autoimmune diseases develop when the immune system responds adversely to normal body tissues. Autoimmune disorders may result in damage to body tissues, abnormal organ growth and/or changes in organ function. The disorder may affect only one organ or tissue type or may affect multiple organs and tissues. Organs and tissues commonly affected by autoimmune disorders include blood components such as red blood cells, blood vessels, connective tissues, endocrine glands such as the thyroid or pancreas, muscles, joints and skin. An inflammatory disease is a disease characterised by inflammation. Many inflammatory diseases are autoimmune diseases and vice-versa.

[0237] The chronic inflammatory bowel diseases (IBD) Crohn's disease and ulcerative colitis, which afflict both children and adults, are examples of autoimmune and inflammatory diseases of the GIT (Hendrickson et al 2002 Clin Microbiol Rev 15(1):79-94, herein incorporated by reference in its entirety). Ulcerative colitis is defined as a condition where the inflammatory response and morphologic changes remain confined to the colon. The rectum is involved in 95% of patients. Inflammation is largely limited to the mucosa and consists of continuous involvement of variable severity with ulceration, edema, and hemorrhage along the length of the colon (Hendrickson et al 2002 Clin. Microbiol Rev 15(1):79-94, herein incorporated by reference in its entirety). Ulcerative colitis is usually manifested by the presence of blood and mucus mixed with stool, along with lower abdominal cramping which is most severe during the passage of bowel movements. Clinically, the presence of diarrhoea with blood and mucus differentiates ulcerative colitis from irritable bowel syndrome, in which blood is absent. Unlike ulcerative colitis, the presentation of Crohn's disease is usually subtle, which leads to a later diagnosis. Factors such as the location, extent, and severity of involvement determine the extent of gastrointestinal symptoms. Patients who have ileocolonic involvement usually have postprandial abdominal pain, with tenderness in the right lower quadrant and an occasional inflammatory mass.

[0238] Suitably the pharmaceutical composition or construct of the invention is for use in the treatment of an autoimmune and/or inflammatory disease of the GIT selected from the list consisting of Crohn's disease, ulcerative colitis, irritable bowel syndrome, diabetes type II, glomerulonephritis, autoimmune hepatitis, Sjogren's syndrome, celiac disease and drug- or radiation-induced mucositis (most suitably Crohn's disease).

Infection of the GIT

[0239] Viral, bacterial, parasitic and other pathogenic infections can occur in the GIT. These may be confined to the GIT or initiated in the GIT before spreading to other parts of the body. The construct of the invention may be used for the treatment or prevention of bacterial infection including infection by common bacterial GIT pathogens including Escherichia coli, Salmonella, Campylobacter, Vibrio cholerae, Shigella, Clostridium perfringens, Clostridium difficile, Bacillus cereus, Vibrio parahaemolyticus, Yersinia enerocolitica. The construct of the invention may be used for the treatment or prevention of viral infection including common viral GIT pathogens which include rotavirus, norovirus and small round viruses. Suitably the construct of the invention is for use in the treatment or prevention of nosocomial infection. Suitably the construct of the invention is for use in the treatment or prevention of C. difficile infection.

[0240] Suitably, the first and/or second polypeptide of the construct of the invention binds to a target accessible via the intestinal tract such as a target within the intestinal tract. Suitably the target is a deleterious agent originating from an intestinal tract resident pathogenic microbe. Suitably the target is selected from the group consisting of: an interleukin (such as IL-1, IL-1ra, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18 and IL-23), an interleukin receptor (such as IL-6R and IL-7R), a transcription factor (such as NF-kB), a cytokine (such as TNF-alpha, IFN-gamma TGF-beta and TSLP), a transmembrane protein (such as gp130 and CD3), a surface glycoprotein (such as CD4, CD20, CD40), a soluble protein (such as CD40L), an integrin (such as a4b7 and AlphaEbeta7), an adhesion molecule (such as MAdCAM), a chemokine (such as IP10 and CCL20), a chemokine receptor (such as CCR2 and CCR9), an inhibitory protein (SMAD7), a kinase (such as JAK3), a G protein-coupled receptor (such as sphingosine-1-P receptor) and a toxin (such as C. difficile toxin A and C. difficile toxin B); more suitably the target is selected from the group consisting of: TNF-alpha, C. difficile toxin A, C. difficile toxin B, CD3 and IL-6R; more suitably TNF-alpha or C. difficile toxin A. Suitably the target of the first and second polypeptides are identical or different.

[0241] In one embodiment the first and/or second polypeptide of the construct of the invention (such as the first and/or second immunoglobulin chain variable domain) does not bind to an interleukin (such as IL-1, IL-1ra, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18 and IL-23).

[0242] In a further embodiment the first and/or second polypeptide of the construct of the invention (such as the first and/or second immunoglobulin chain variable domain) does not bind to an interleukin receptor (such as IL-6R and IL-7R).

[0243] In a further embodiment the first and/or second polypeptide of the construct of the invention (such as the first and/or second immunoglobulin chain variable domain) does not bind to a transcription factor (such as NF-kB).

[0244] In a further embodiment the first and/or second polypeptide of the construct of the invention (such as the first and/or second immunoglobulin chain variable domain) does not bind to a cytokine (such as TNF-alpha, IFN-gamma TGF-beta and TSLP).

[0245] In a further embodiment the first and/or second polypeptide of the construct of the invention (such as the first and/or second immunoglobulin chain variable domain) does not bind to a transmembrane protein (such as gp130 and CD3).

[0246] In a further embodiment the first and/or second polypeptide of the construct of the invention (such as the first and/or second immunoglobulin chain variable domain) does not bind to a surface glycoprotein (such as CD4, CD20 and CD40).

[0247] In a further embodiment the first and/or second polypeptide of the construct of the invention (such as the first and/or second immunoglobulin chain variable domain) does not bind to a soluble protein (such as CD40L).

[0248] In a further embodiment the first and/or second polypeptide of the construct of the invention (such as the first and/or second immunoglobulin chain variable domain) does not bind to an integrin (such as a4b7 and AlphaEbeta7).

[0249] In a further embodiment the first and/or second polypeptide of the construct of the invention (such as the first and/or second immunoglobulin chain variable domain) does not bind to an adhesion molecule (such as MAdCAM)

[0250] In a further embodiment the first and/or second polypeptide of the construct of the invention (such as the first and/or second immunoglobulin chain variable domain) does not bind to a chemokine (such as IP10 and CCL20).

[0251] In a further embodiment the first and/or second polypeptide of the construct of the invention (such as the first and/or second immunoglobulin chain variable domain) does not bind to a chemokine receptor (such as CCR2 and CCR9).

[0252] In a further embodiment the first and/or second polypeptide of the construct of the invention (such as the first and/or second immunoglobulin chain variable domain) does not bind to an inhibitory protein (SMAD7).

[0253] In a further embodiment the first and/or second polypeptide of the construct of the invention (such as the first and/or second immunoglobulin chain variable domain) does not bind to a kinase (such as JAK3).

[0254] In a further embodiment the first and/or second polypeptide of the construct of the invention (such as the first and/or second immunoglobulin chain variable domain) does not bind to a G protein-coupled receptor (such as sphingosine-1-P receptor).

[0255] In a further embodiment the first and/or second polypeptide of the construct of the invention (such as the first and/or second immunoglobulin chain variable domain) does not bind to a toxin (such as C. difficile toxin A and C. difficile toxin B).

Therapeutic Use and Delivery

[0256] Suitably the construct or composition of the invention is for use as a medicament, suitably administered by oral administration, suitably for use in the treatment or prevention of diseases of the GIT (see intra). The construct or composition of the invention may also be used in the treatment or prevention of other medical conditions by oral administration such as metabolic disorders, such as obesity. In one embodiment, the construct or composition of the invention is intended to have local effect in the intestinal tract. In one embodiment, the construct or composition of the invention is not for use in the treatment or prevention of diseases by delivery into the circulation in therapeutically effective quantities.

[0257] In one aspect of the invention there is provided a method of treating autoimmune disease or C. difficile infection comprising administering to a person in need thereof a therapeutically effective amount of the inventive construct or composition.

[0258] A therapeutically effective amount of a construct of the invention is an amount which is effective, upon single or multiple dose administration to a subject, in neutralising the biological effects of a chosen target to a significant extent in a subject. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the construct to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the construct are outweighed by the therapeutically beneficial effects. The construct of the invention can be incorporated into pharmaceutical compositions suitable for oral administration to a subject. The construct of the invention can be in the form of a pharmaceutically acceptable salt.

[0259] In one aspect of the invention there is provided a method of treating a disease with a monomeric antibody or a monomeric antigen binding fragment thereof, comprising administering to a subject the inventive construct. There is also provided a method of treating a disease with two or more monomeric antibodies or monomeric antigen binding fragments thereof, comprising administering to a subject the inventive construct.

[0260] A pharmaceutical composition of the invention is formulated for oral delivery. The pharmaceutical compositions of the invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions, dispersions or suspensions, tablets, pills and powders. Solid dosage forms are preferred. The pharmaceutical composition may comprise a pharmaceutically acceptable excipient, and suitably may be used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0261] Typically, the pharmaceutical composition comprises a construct of the invention and a pharmaceutically acceptable excipient such as a carrier. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the polypeptide or construct of the invention. Pharmaceutical compositions may include antiadherents, binders, coatings, disintegrants, flavours, colours, lubricants, sorbents, preservatives, sweeteners, freeze dry excipients (including lyoprotectants) or compression aids. Suitably, the construct of the invention is lyophilised before being incorporated into a pharmaceutical composition.

[0262] Suitably, the first and second polypeptides of the construct are substantially resistant to all proteases present in the intestinal tract by virtue of the inherent properties of the polypeptides or construct itself.

[0263] A polypeptide of the invention may also be provided with an enteric coating. An enteric coating is a polymer barrier applied on oral medication which protects the polypeptide from the low pH of the stomach. Materials used for enteric coatings include fatty acids, waxes, shellac, plastics, and plant fibers. Suitable enteric coating components include methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, sodium alginate and stearic acid. Suitable enteric coatings include pH-dependent release polymers. These are polymers which are insoluble at the highly acidic pH found in the stomach, but which dissolve rapidly at a less acidic pH. Thus, suitably, the enteric coating will not dissolve in the acidic juices of the stomach (pH .about.3), but will do so in the higher pH environment present in the small intestine (pH above 6) or in the colon (pH above 7.0). The pH-dependent release polymer is selected such that the construct of the invention will be released at about the time that the dosage reaches the target region of the intestinal tract.

[0264] The pharmaceutical composition or construct of the invention may be formulated in a buffer, in order to stabilise the pH of the composition, at a concentration between 5-50, or more suitably 15-40 or more suitably 25-30 g/litre. Examples of suitable buffer components include physiological salts such as sodium citrate and/or citric acid. Suitably buffers contain 100-200, more suitably 125-175 mM physiological salts such as sodium chloride. Suitably the buffer is selected to have a pKa close to the pH of the composition or the physiological pH of the patient.

[0265] Exemplary construct concentrations in a pharmaceutical composition may range from about 1 mg/mL to about 200 mg/ml or from about 50 mg/mL to about 200 mg/mL, or from about 150 mg/mL to about 200 mg/mL.

[0266] An aqueous formulation of the construct or pharmaceutical composition of the invention may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of suitable buffers include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, for example, on the buffer and the desired tonicity of the formulation.

[0267] The tonicity of the pharmaceutical composition may be altered by including a tonicity modifier. Such tonicity modifiers can be charged or uncharged chemical species. Typical uncharged tonicity modifiers include sugars or sugar alcohols or other polyols, preferably trehalose, sucrose, mannitol, glycerol, 1,2-propanediol, raffinose, sorbitol or lactitol (especially trehalose, mannitol, glycerol or 1,2-propanediol). Typical charged tonicity modifiers include salts such as a combination of sodium, potassium or calcium ions, with chloride, sulfate, carbonate, sulfite, nitrate, lactate, succinate, acetate or maleate ions (especially sodium chloride or sodium sulphate); or amino acids such as arginine or histidine. Suitably, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 1 mM to 500 nM. Suitably, at least one isotonic agent is included in the composition.

[0268] A surfactant may also be added to the pharmaceutical composition to reduce aggregation of the formulated construct and/or minimize the formation of particulates in the formulation and/or reduce adsorption. Exemplary surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, and polysorbate 80. Exemplary concentrations of surfactant may range from about 0.001% to about 10% w/v.

[0269] A lyoprotectant may also be added in order to protect the construct of the invention against destabilizing conditions during the lyophilization process. For example, known lyoprotectants include sugars (including glucose, sucrose, mannose and trehalose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included in an amount of about 10 mM to 500 mM.

[0270] The dosage ranges for administration of the pharmaceutical composition or construct of the invention are those to produce the desired therapeutic effect. The dosage range required depends on the precise nature of the pharmaceutical composition or construct, the target region of the intestinal tract, the nature of the formulation, the age of the patient, the nature, extent or severity of the patients condition, contraindications, if any, and the judgement of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation.

[0271] Suitable daily dosages of pharmaceutical composition or construct of the invention are in the range of 50 ng-50 mg per kg, such as 50 ug-40 mg per kg, such as 5-30 mg per kg of body weight. The unit dosage can vary from less than 100 mg, but typically will be in the region of 250-2000 mg per dose, which may be administered daily or more frequently, for example 2, 3 or 4 times per day or less frequently for example every other day or once per week.

[0272] Treatment of diseases also embraces treatment of exacerbations thereof and also embraces treatment of patients in remission from disease symptoms to prevent relapse of disease symptoms.

Combination Therapy

[0273] A pharmaceutical composition of the invention may also comprise one or more active agents (e.g. active agents suitable for treating diseases such as those mentioned herein). It is within the scope of the invention to use the pharmaceutical composition of the invention in therapeutic methods for the treatment of bacterial infection, autoimmune and/or inflammatory diseases as an adjunct to, or in conjunction with, other established therapies normally used in the treatment of bacterial, autoimmune and/or inflammatory diseases.

[0274] For the treatment of irritable bowel disease (such as Crohn's disease or ulcerative colitis), possible combinations include combinations with, for example, one or more active agents selected from the list comprising: 5-aminosalicylic acid, or a prodrug thereof (such as sulfasalazine, olsalazine or bisalazide); corticosteroids (e.g. prednisolone, methylprednisolone, or budesonide); immunosuppressants (e.g. cyclosporin, tacrolimus, methotrexate, azathioprine or 6-mercaptopurine); anti-TNF-alpha antibodies (e.g., infliximab, adalimumab, certolizumab pegol or golimumab); anti-IL12/1L23 antibodies (e.g., ustekinumab); anti-IL6R antibodies or small molecule IL12/1L23 inhibitors (e.g., apilimod); Anti-alpha-4-beta-7 antibodies (e.g., vedolizumab); MAdCAM-1 blockers (e.g., PF-00547659); antibodies against the cell adhesion molecule alpha-4-integrin (e.g., natalizumab); antibodies against the IL2 receptor alpha subunit (e.g., daclizumab or basiliximab); JAK3 inhibitors (e.g., tofacitinib or R348); Syk inhibitors and prodrugs thereof (e.g., fostamatinib and R-406); Phosphodiesterase-4 inhibitors (e.g., tetomilast); HMPL-004; probiotics; Dersalazine; semapimod/CPSI-2364; and protein kinase C inhibitors (e.g. AEB-071). The most suitable combination agents are infliximab, adalimumab, certolizumab pegol or golimumab.

[0275] For the treatment of bacterial infections, such as Clostridium difficile infection, possible combinations include combinations with, for example, one or more active agents selected from the list comprising C. difficile toxoid vaccine, ampicillin, amoxicillin, vancomycin, metronidazole, fidaxomicin, linezolid, nitazoxanide, rifaximin, ramoplanin, difimicin, clindamycin, cephalosporins (such as second and third generation cephalosporins), fluoroquinolones (such as gatifloxacin or moxifloxacin), macrolides (such as erythromycin, clarithromycin, azithromycin), penicillins, aminoglycosides, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, imipenem, meropenem, antibacterial agents, bactericides, or bacteriostats. Possible combinations also include combinations with one or more active agents which are probiotics, for example Saccharomyces boulardii or Lactobacillus rhamnosus GG.

[0276] Hence another aspect of the invention provides a pharmaceutical composition of the invention in combination with one or more further active agents, for example one or more active agents described above. In a further aspect of the invention, the pharmaceutical composition or construct is administered sequentially, simultaneously or separately with at least one active agent selected from the list above.

[0277] Similarly, another aspect of the invention provides a combination product comprising: [0278] (A) a pharmaceutical composition or construct of the present invention; and [0279] (B) one or more other active agents, wherein each of components (A) and (B) is formulated in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier. In this aspect of the invention, the combination product may be either a single (combination) formulation or a kit-of-parts. Thus, this aspect of the invention encompasses a combination formulation including a pharmaceutical composition or construct of the present invention and another therapeutic agent, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0280] The invention also encompasses a kit of parts comprising components: [0281] (i) a pharmaceutical composition or construct of the present invention in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier; and [0282] (ii) a formulation including one or more other active agents, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier, which components (i) and (ii) are each provided in a form that is suitable for administration in conjunction with the other.

[0283] Component (i) of the kit of parts is thus component (A) above in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier. Similarly, component (ii) is component (B) above in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier. The one or more other active agents (i.e. component (B) above) may be, for example, any of the agents mentioned above in connection with the treatment of bacterial infection such as Clostridium difficile infection, autoimmune and/or inflammatory diseases such as IBD (e.g. Crohn's disease and/or ulcerative colitis). If component (B) is more than one further active agent, these further active agents can be formulated with each other or formulated with component (A) or they may be formulated separately. In one embodiment component (B) is one other therapeutic agent. In another embodiment component (B) is two other therapeutic agents. The combination product (either a combined preparation or kit-of-parts) of this aspect of the invention may be used in the treatment or prevention of an autoimmune disease (e.g. the autoimmune diseases mentioned herein).

Vectors and Hosts

[0284] The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a plasmid, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian and yeast vectors). Other vectors (e.g. non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g. replication defective retroviruses. adenoviruses and adeno-associated viruses), which serve equivalent functions, and also bacteriophage and phagemid systems. The invention also relates to nucleotide sequences that encode constructs of the invention. The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. Such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell.

[0285] In one aspect of the invention there is provided a vector comprising the polynucleotide encoding the construct of the invention or cDNA comprising said polynucleotide. In a further aspect of the invention there is provided a host cell transformed with said vector, which is capable of expressing the construct of the invention. Suitably the host cell is a yeast such as a yeast belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia, such as Saccharomyces cerevisiae, Escherchia coli or Pichia pastoris.

[0286] It is particularly advantageous for production and convenience purposes if the labile peptide linker is cleaved by proteases present in the intestinal tract, but if the labile peptide linker is also substantially resistant to proteases of the host organism in which the construct is produced. Therefore, in one embodiment of the invention, the labile peptide linker is substantially resistant to proteases of the host organism in which the construct is produced.

Preparative Methods

[0287] Constructs of the invention can be obtained and manipulated using the techniques disclosed for example in Green and Sambrook 2012 Molecular Cloning: A Laboratory Manual 4.sup.th Edition Cold Spring Harbour Laboratory Press.

[0288] In particular, artificial gene synthesis may be used to produce a construct according to the invention (Nambiar et al 1984 Science 223:1299-1301, Sakamar and Khorana 1988 Nucl. Acids Res 14:6361-6372, Wells et al 1985 Gene 34:315-323 and Grundstrom et al 1985 Nucl. Acids Res 13:3305-3316, herein incorporated by reference in their entirety). A gene encoding a construct of the invention can be synthetically produced by, for example, solid-phase DNA synthesis. Entire genes may be synthesized de novo, without the need for precursor template DNA. To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product. Upon the completion of the chain assembly, the product is released from the solid phase to solution, deprotected, and collected. Products can be isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotides in high purity (Verma and Eckstein 1998 Annu Rev Biochem 67:99-134).

[0289] The constructs of the invention may be fused genetically at the DNA level i.e. a polynucleotide construct which encodes the complete polypeptide construct comprising one or more polypeptides such as antigen-binding polypeptides. One way of joining multiple polypeptides via the genetic route is by linking the polypeptide coding sequences via a labile peptide linker coding sequence. For example, the carboxy-terminal end of the first polypeptide may be linked to the amino-terminal end of the next polypeptide via a labile peptide linker coding sequence. This linking mode can be extended in order to link polypeptides for the construction of tri-, tetra-, etc. functional constructs. A method for producing multivalent (such as bivalent) VHH polypeptide constructs is disclosed in WO96/34103 (herein incorporated by reference in its entirety).

[0290] Mutations can be made to the DNA or cDNA that encode constructs which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli and S. cerevisiae, are known.

[0291] Mutation of constructs can be achieved for example by substitutions, additions or deletions to a nucleic acid encoding the construct. The substitutions, additions or deletions to a nucleic acid encoding the construct can be introduced by many methods, including for example error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis (Ling et al 1997 Anal Biochem 254(2):157-178, herein incorporated by reference in its entirety), gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR) or a combination of these methods. The modifications, additions or deletions to a nucleic acid can also be introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, ensemble mutagenesis, chimeric nucleic acid multimer creation, or a combination thereof.

[0292] Expression of constructs comprising immunoglobulin chain variable domains such as VHs and VHHs can be achieved using a suitable expression vector such as a prokaryotic cell such as bacteria, for example E. coli (for example according to the protocols disclosed in WO94/04678 and WO96/34103, which are incorporated herein by reference). Expression of immunoglobulin chain variable domains such as VHs and VHHs can also be achieved using eukaryotic cells, for example insect cells, CHO cells, Vero cells or suitably yeast cells such as yeasts belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia. Suitably S. cerevisiae is used (for example according to the protocols disclosed in WO94/025591, which is incorporated herein by reference).

[0293] Suitably, the construct of the invention can be produced in a fungus such as a yeast (for example, S. cerevisiae) comprising growth of the fungus on a medium comprising a carbon source wherein 50-100 wt % of said carbon source is ethanol, according to the methods disclosed in WO02/48382.

[0294] In one aspect of the invention there is provided a process for the preparation of the construct of the invention comprising the following steps: [0295] i) cloning into a vector, such as a plasmid, the polynucleotide of the invention, [0296] ii) transforming a cell, such as a bacterial cell or a yeast cell capable of producing the construct of the invention, with said vector in conditions allowing the production of the construct, [0297] iii) recovering the construct, such as by affinity chromatography.

Clauses

[0298] A set of clauses defining the invention and its preferred aspects is as follows: [0299] 1. A construct suitable for oral administration comprising a first polypeptide and a second polypeptide connected by a labile peptide linker, wherein the labile peptide linker is labile to one or more proteases present in the intestinal tract and wherein the first and second polypeptides are substantially resistant to said one or more proteases. [0300] 2. The construct according to clause 1, wherein the first polypeptide is an antigen-binding polypeptide. [0301] 3. The construct according to clause 2, wherein the antigen-binding polypeptide is an antibody or an antigen-binding fragment thereof. [0302] 4. The construct according to any one of clauses 1 to 3, wherein the second polypeptide is an antigen-binding polypeptide. [0303] 5. The construct according to clause 4, wherein the second antigen-binding polypeptide is an antibody or an antigen-binding fragment thereof. [0304] 6. The construct according to any one of clauses 1 to 5 wherein the first and second polypeptides are substantially resistant to all proteases present in the intestinal tract. [0305] 7. The construct according to any one of clauses 1 to 6 wherein the intestinal tract is a mammalian intestinal tract, such as a human, simian, murine, bovine, ovine or porcine intestinal tract. [0306] 8. The construct according to any one of clauses 1 to 7, wherein the one or more proteases are present in the small or large intestine. [0307] 9. The construct according to clause 8, wherein the one or more proteases are present in the jejunum, the ileum and/or the cecum. [0308] 10. The construct according to either clause 8 or 9, wherein the one or more proteases are serine proteases. [0309] 11. The construct according to any one of clauses 1 to 10, wherein the labile peptide linker is substantially resistant to proteases of the host organism in which the construct is produced. [0310] 12. The construct according to any one of clauses 1 to 11, wherein the labile peptide linker comprises a cleavage site for trypsin or a trypsin-like protease. [0311] 13. The construct according to clause 12, wherein the labile peptide linker comprises at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 K residues. [0312] 14. The construct according to any one of clauses 1 to 13, wherein the labile peptide linker comprises at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 R residues. [0313] 15. The construct according to any one of clauses 1 to 14, wherein the labile peptide linker does not comprise any P residues. [0314] 16. The construct according to any one of clauses 1 to 15, wherein all K or R residues comprised within the labile peptide linker have at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5 shielding residues on their N-terminal side, wherein the shielding residues are selected from the list consisting of: D and E. [0315] 17. The construct according to any one of clauses 1 to 16, wherein all K or R residues comprised within the labile peptide linker have at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5 shielding residues on their C-terminal side, wherein the shielding residues are selected from the list consisting of: D and E. [0316] 18. The construct according to either clause 16 or 17, wherein all K and R residues have at least one shielding residue adjacent to them. [0317] 19. The construct according to any one of clauses 1 to 18, wherein the labile peptide linker does not comprise any D or E residues. [0318] 20. The construct according to any one of clauses 1 to 18, wherein the labile peptide linker consists of residues selected from the list consisting of C, A, S, N, G, L, I, V, T, M, F, Y, H, K, R, W and Q. [0319] 21. The construct according to clause 20, wherein the labile peptide linker consists of residues selected from the list consisting of A, G, L, I, V, M, S, T, K and R residues. [0320] 22. The construct according to clause 21, wherein the labile peptide linker consists of residues selected from the list consisting of S, G, K and R. [0321] 23. The construct according to any one of clauses 1 to 15, wherein all K or R residues comprised within the labile peptide linker have at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5 non-shielding residues on their N-terminal side, wherein the non-shielding residues are selected from the list consisting of: C, A, S, N, G, L, I, V, T, M, F, Y, H, W and Q. [0322] 24. The construct according to any one of clauses 1 to 15 and 23, wherein all K or R residues comprised within the labile peptide linker have at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5 non-shielding residues on their C-terminal side, wherein the non-shielding residues are selected from the list consisting of: C, A, S, N, G, L, I, V, T, M, F, Y, H, W and Q. [0323] 25. The construct according to either clause 23 or 24, wherein all K and R residues have at least one non-shielding residue adjacent to them. [0324] 26. The construct according to any one of clauses 23 to 25, wherein the non-shielding residues are selected from the list consisting of: A, G, L, I, V, M, S and T. [0325] 27. The construct according to clause 26, wherein the non-shielding residues are selected from the list consisting of: A, G, L, I, V and S. [0326] 28. The construct according to clause 27, wherein the non-shielding residues are selected from the list consisting of: G and S. [0327] 29. The construct according to any one of clauses 1 to 11, wherein the labile peptide linker comprises a cleavage site for chymotrypsin or a chymotrypsin-like protease. [0328] 30. The construct according to clause 29, wherein the labile peptide linker comprises at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 residues selected from the list consisting of W, F, Y, L and M. [0329] 31. The construct according to clause 30, wherein the labile peptide linker comprises at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 residues selected from the list consisting of W, F and Y. [0330] 32. The construct according to any one of clauses 29 to 31, wherein the labile peptide linker consists of residues selected from the list consisting of S, G, W, F, Y, L and M; such as S, G, W, F and Y. [0331] 33. The construct according to any one of clauses 1 to 32, wherein the labile peptide linker has a length of at least 3 residues, such as at least 5 residues, such as at least 10 residues. [0332] 34. The construct according to any one of clauses 1 to 33, wherein the labile peptide linker has a length of no greater than 40 residues, such as no greater than 25 residues, such as no greater than 15 residues. [0333] 35. The construct according to clause 1, wherein the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:

[0333] [-(G.sub.aS).sub.x--U.sub.c--B--U'.sub.d-(G.sub.bS).sub.y--].sub.- z

wherein [0334] a is 1 to 10; [0335] b is 1 to 10; [0336] U is D or E; [0337] U' is D or E; [0338] c is 0 to 7; [0339] d is 0 to 7; [0340] x is 1 to 10; [0341] y is 1 to 10 [0342] z is 1 to 10 and [0343] B is K or R. [0344] 36. The construct according to clause 35, wherein the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:

[0344] [-(G.sub.aS).sub.x--B-(G.sub.bS).sub.y--].sub.z [0345] wherein [0346] a is 1 to 10; [0347] b is 1 to 10; [0348] x is 1 to 10; [0349] y is 1 to 10 [0350] z is 1 to 10 and [0351] B is K or R. [0352] 37. The construct according to clause 36, wherein the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:

[0352] [-(G.sub.4S).sub.x--B-(G.sub.4S).sub.y--].sub.z [0353] wherein [0354] x is 1 to 10; [0355] y is 1 to 10 [0356] z is 1 to 10 and [0357] B is K or R. [0358] 38. The construct according to clause 1, wherein the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:

[0358] --B-(G.sub.aS).sub.x--B'-- [0359] wherein [0360] a is 1 to 10; [0361] x is 1 to 10; [0362] B is K or R and [0363] B' is K or R. [0364] 39. The construct according to clause 1, wherein the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:

[0364] --B-(G.sub.aS).sub.x--B'-(G.sub.bS).sub.y--B''-- [0365] wherein [0366] a is 1 to 10; [0367] b is 1 to 10; [0368] x is 1 to 10; [0369] y is 1 to 10; [0370] B is K or R [0371] B' is K or R and [0372] B'' is K or R. [0373] 40. The construct according to any one of clauses 35 to 39, wherein a is 2 to 5. [0374] 41. The construct according to clause 40, wherein a is 4. [0375] 42. The construct according to any one of clauses 35 to 41, wherein b is 2 to 5. [0376] 43. The construct according to clause 42, wherein b is 4. [0377] 44. The construct according to any one of clauses 35 to 43, wherein x is 1 to 5. [0378] 45. The construct according to clause 44, wherein x is 2. [0379] 46. The construct according to clause 44, wherein x is 1. [0380] 47. The construct according to any one of clauses 35 to 46, wherein y is 1 to 5. [0381] 48. The construct according to clause 47, wherein y is 1. [0382] 49. The construct according to clause 47, wherein y is 2. [0383] 50. The construct according to any one of clauses 35 to 49, wherein z is 1 to 3. [0384] 51. The construct according to clause 50, wherein z is 1. [0385] 52. The construct according to any one of clauses 35 to 51, wherein B is K. [0386] 53. The construct according to any one of clauses 35 to 52, wherein U is D. [0387] 54. The construct according to any one of clauses 35 to 53, wherein U' is D. [0388] 55. The construct according to any one of clauses 35 to 54, wherein c is 1. [0389] 56. The construct according to any one of clauses 35 to 55, wherein d is 1. [0390] 57. The construct according to any one of clauses 35 to 54, wherein c is 0. [0391] 58. The construct according to any one of clauses 35 to 55 or 57, wherein d is 0. [0392] 59. The construct according to any one of clauses 35 to 54, wherein c is 4 and d is 0. [0393] 60. The construct according to any one of clauses 35 to 59, wherein B' is K. [0394] 61. The construct according to any one of clauses 35 to 60, wherein B'' is K. [0395] 62. The construct according to clause 1, wherein the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:

[0395] [-(G.sub.aS).sub.x-J-(G.sub.bS).sub.y--].sub.z [0396] wherein [0397] a is 1 to 10; [0398] b is 1 to 10; [0399] x is 1 to 10; [0400] y is 1 to 10 [0401] z is 1 to 10 and [0402] J is W, F, Y, L or M. [0403] 63. The construct according to clause 62, wherein the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:

[0403] [-(G.sub.4S).sub.x-J-(G.sub.4S).sub.y--].sub.z [0404] wherein [0405] x is 1 to 10; [0406] y is 1 to 10 [0407] z is 1 to 10 and [0408] J is W, F, Y, L or M. [0409] 64. The construct according to clause 1, wherein the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format: [0410] wherein [0411] a is 1 to 10; [0412] x is 1 to 10; [0413] J is W, F, Y, L or M and [0414] J' is W, F, Y, L or M. [0415] 65. The construct according to clause 1, wherein the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:

[0415] -J-(G.sub.aS).sub.x-J'-(G.sub.bS).sub.y-J''-- [0416] wherein [0417] a is 1 to 10; [0418] b is 1 to 10; [0419] x is 1 to 10; [0420] y is 1 to 10; [0421] J is W, F, Y, L or M [0422] J' is W, F, Y, L or M and [0423] J'' is W, F, Y, L or M. [0424] 66. The construct according to any one of clauses 62 to 65, wherein a is 2 to 5. [0425] 67. The construct according to clause 66, wherein a is 4. [0426] 68. The construct according to any one of clauses 62 to 67, wherein b is 2 to 5. [0427] 69. The construct according to clause 68, wherein b is 4. [0428] 70. The construct according to any one of clauses 62 to 69, wherein x is 1 to 5. [0429] 71. The construct according to clause 70, wherein x is 2. [0430] 72. The construct according to clause 70, wherein x is 1. [0431] 73. The construct according to any one of clauses 62 to 72, wherein y is 1 to 5. [0432] 74. The construct according to clause 73, wherein y is 1. [0433] 75. The construct according to clause 73, wherein y is 2. [0434] 76. The construct according to any one of clauses 62 to 75, wherein z is 1 to 3. [0435] 77. The construct according to clause 76, wherein z is 1. [0436] 78. The construct according to any one of clauses 62 to 77, wherein J is W, F or Y. [0437] 79. The construct according to any one of clauses 62 to 78, wherein J' is W, F or Y. [0438] 80. The construct according to any one of clauses 62 to 79, wherein J'' is W, F or Y. [0439] 81. The construct according to any one of clauses 1 to 80, wherein the first antibody or antigen binding fragment thereof binds to a first target accessible via the intestinal tract, such as a target within the intestinal tract. [0440] 82. The construct according to clause 81, wherein the first target is a first deleterious agent originating from an intestinal tract resident pathogenic microbe. [0441] 83. The construct according to clause 81, wherein the first target is selected from the group consisting of: TNF-alpha, C. difficile toxin A, C. difficile toxin B, CD3 or IL-6R. [0442] 84. The construct according to clause 83, wherein the first target is selected from the group consisting of: TNF-alpha or C. difficile toxin A. [0443] 85. The construct according to clause 81, wherein the second antibody or antigen binding fragment thereof binds to a second target accessible via the intestinal tract, such as a target within the intestinal tract. [0444] 86. The construct according to clause 85, wherein second target is a second deleterious agent originating from an intestinal tract resident pathogenic microbe. [0445] 87. The construct according to clause 85, wherein the second target is selected from the group consisting of: TNF-alpha, C. difficile toxin A, C. difficile toxin B, CD3 or IL-6R. [0446] 88. The construct according to clause 87, wherein the second target is selected from the group consisting of: TNF-alpha or C. difficile toxin A. [0447] 89. The construct according to any one of clauses 81 to 88, wherein the first and second targets are identical. [0448] 90. The construct according to any one of clauses 81 to 88, wherein the first and second targets are different. [0449] 91. The construct according to any one of clauses 1 to 90, wherein the antigen-binding fragments are selected from the group consisting of: VLs, V-NARs, scFvs, Fab fragments, F(ab')2 fragments or immunoglobulin chain variable domains such as VHHs and VHs. [0450] 92. The construct according to clause 91, wherein the antigen-binding fragments are VHHs. [0451] 93. The construct according to clause 91, wherein the antigen-binding fragments are VHs. [0452] 94. The construct according to any one of clauses 1 to 93, wherein at least 20%, such as at least 40%, such as at least 60%, such as at least 80%, such as at least 90%, such as at least 100% by mass of the construct remains uncleaved after at least 10, such as at least 20, such as at least 30 minutes after mixing in the Faecal Protease Assay and/or Trypsin Protease Assay and/or Chymotrypsin Protease Assay. [0453] 95. The construct according to clause 1, wherein the first and second polypeptides each comprise a sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 19. [0454] 96. The construct according to clause 95 wherein the first and second polypeptides each consist of a sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 19. [0455] 97. The construct according to clause 1 wherein the construct comprises a sequence selected from the group consisting of SEQ ID NOs: 7 to 17. [0456] 98. The construct according to clause 97 wherein the construct consists of a sequence selected from the group consisting of SEQ ID NOs: 7 to 17. [0457] 99. The construct according to clause 1 wherein the labile peptide linker comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 6. [0458] 100. The construct according to clause 99 wherein the labile peptide linker consists of a sequence selected from the group consisting of SEQ ID NOs: 1 to 6. [0459] 101. The construct according to any one of clauses 1 to 94, wherein the construct comprises an additional third polypeptide connected by a peptide linker to the first polypeptide, wherein the third polypeptide is substantially resistant to the one or more proteases present in the intestinal tract. [0460] 102. The construct according to clause 101, wherein the construct comprises or consists of an additional fourth polypeptide connected by a peptide linker to the second polypeptide, wherein the fourth polypeptide is substantially resistant to the one or more proteases present in the intestinal tract. [0461] 103. The construct according to clause 101 or 102, wherein the peptide linkers are substantially resistant to the one or more proteases present in the intestinal tract. [0462] 104. The construct according to clause 101 or 102, wherein the peptide linkers are labile peptide linkers. [0463] 105. The construct according to any one of clauses 1 to 104, wherein the construct is enterically-coated. [0464] 106. A pharmaceutical composition comprising a construct according to any one of clauses 1 to 105 and a pharmaceutically acceptable excipient. [0465] 107. The construct or composition according to any one of clauses 1 to 106 for use as a medicament. [0466] 108. The construct or composition according to clause 107 wherein the medicament is administered by oral administration. [0467] 109. The construct or composition according to clause 108 for use in the treatment of diseases of the GIT. [0468] 110. The construct or composition according to clause 109 for use in the treatment of autoimmune and/or inflammatory disease. [0469] 111. The construct or composition according to clause 110 for use in the treatment of inflammatory bowel disease such as Crohn's disease and/or ulcerative colitis. [0470] 112. The construct or composition according to clause 109 for use in the treatment of bacterial infection such as C. difficile infection. [0471] 113. A nucleic acid comprising a sequence encoding the construct according to any one of clauses 1 to 104. [0472] 114. A method of treating autoimmune disease comprising administering to a person in need thereof a therapeutically effective amount of a construct or composition according to any one of clauses 1 to 106. [0473] 115. A method of treating bacterial infection such as C. difficile infection comprising administering to a person in need thereof a therapeutically effective amount of a construct or composition according to any one of clauses 1 to 106. [0474] 116. A method of treating a disease with a monomeric polypeptide, comprising administering to a subject the construct or composition of any one of clauses 1 to 106. [0475] 117. A method of treating a disease with two or more monomeric polypeptides, comprising administering to a subject the construct or composition of any one of clauses 1 to 106. [0476] 118. A method of treating two or more diseases with two or more monomeric polypeptides, comprising administering to a subject the construct or composition of any one of clauses 1 to 106. [0477] 119. The method of any one of clauses 114 to 118 wherein the disease is an autoimmune disease and/or inflammatory disease or GIT infection. [0478] 120. A method of assaying the lability of a construct according to any one of clauses 1 to 104, comprising the steps of: (a) incubating the construct in a solution comprising trypsin, a solution comprising chymotrypsin, faecal supernatant, small intestinal fluid, or a solution comprising enteropeptidase, such as by performing the Trypsin Protease Assay of Example 1, the Chymotrypsin Protease Assay of Example 2 or the Faecal Protease Assay of Example 3, then (b) ascertaining the proportion of cleaved construct after one or more periods of incubation. [0479] 121. The method according to clause 120, wherein the level of lability is the percentage of the construct remaining uncleaved after at least 10, such as at least 20, such as at least 30 minutes after mixing in the Trypsin Protease Assay of Example 1, the Chymotrypsin Protease Assay of Example 2 or the Faecal Protease Assay of Example 3. [0480] 122. A method of delivering a monomeric antibody or a monomeric antigen binding fragment thereof to a targeted region of the intestinal tract, comprising the steps of: (a) performing the method according to either clauses 120 or 121 then (b) selecting a construct with an appropriate level of lability for the targeted region of the intestinal tract, (c) producing the selected construct with an enterically coated packaging then (d) administering the packaged selected construct to a subject. [0481] 123. A method of delivering a monomeric polypeptide to the intestinal tract, comprising administering to a subject the construct or composition of any one of clauses 1 to 106. [0482] 124. The method of any one of clauses 116 to 119 or 122 to 123 wherein the subject is a mammal such as a human. [0483] 125. A method of preparing a product comprising a construct according to any one of clauses 1 to 104 which has been selected, the method comprising adding the selected construct into the product, wherein the selected construct is selected and produced by a method comprising the steps of: (a) performing the method according to either clause 120 or 121 then (b) selecting a construct with an appropriate level of lability for the targeted region of the intestinal tract. [0484] 126. A polypeptide comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6. [0485] 127. A polynucleotide comprising SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36.

Further Clauses

[0486] A set of further clauses defining the invention and its preferred aspects is as follows: [0487] 1. A construct suitable for oral administration comprising a first polypeptide and a second polypeptide connected by a labile peptide linker, wherein the labile peptide linker is labile to one or more proteases present in the intestinal tract and wherein the first and second polypeptides are substantially resistant to said one or more proteases. [0488] 2. The construct according to clause 1, wherein the first polypeptide is an antigen-binding polypeptide and the second polypeptide is an antigen-binding polypeptide. [0489] 3. The construct according to either clause 1 or 2 wherein the first and second polypeptides are substantially resistant to all proteases present in the intestinal tract. [0490] 4. The construct according to any one of clauses 1 to 3, wherein the labile peptide linker comprises at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 K and/or R residues. [0491] 5. The construct according to any one of clauses 1 to 4, wherein all K or R residues comprised within the labile peptide linker have at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5 shielding residues on their N-terminal and/or C-terminal side, wherein the shielding residues are selected from the list consisting of: D and E. [0492] 6. The construct according to any one of clauses 1 to 4, wherein all K or R residues comprised within the labile peptide linker have at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5 non-shielding residues on their N-terminal and/or C-terminal side, wherein the non-shielding residues are selected from the list consisting of: C, A, S, N, G, L, I, V, T, M, F, Y, H, W and Q. [0493] 7. The construct according to clause 1, wherein the labile peptide linker comprises a polypeptide sequence of the format:

[0493] [-(G.sub.aS).sub.x--U.sub.c--B--U'.sub.d-(G.sub.bS).sub.y--].sub.- z [0494] wherein [0495] a is 1 to 10; [0496] b is 1 to 10; [0497] U is D or E; [0498] U' is D or E; [0499] c is 0 to 7; [0500] d is 0 to 7; [0501] x is 1 to 10; [0502] y is 1 to 10 [0503] z is 1 to 10 and [0504] B is K or R. [0505] 8. The construct according to clause 7, wherein c and d are both 0. [0506] 9. The construct according to clause 1, wherein the labile peptide linker comprises a polypeptide sequence of the format:

[0506] --B-(G.sub.aS).sub.x--B'-- [0507] wherein [0508] a is 1 to 10; [0509] x is 1 to 10; [0510] B is K or R and [0511] B' is K or R. [0512] 10. The construct according to clause 1, wherein the labile peptide linker comprises a polypeptide sequence of the format:

[0512] --B-(G.sub.aS).sub.x--B'-(G.sub.bS).sub.y--B''-- [0513] wherein [0514] a is 1 to 10; [0515] b is 1 to 10; [0516] x is 1 to 10; [0517] y is 1 to 10; [0518] B is K or R; [0519] B' is K or R and [0520] B'' is K or R. [0521] 11. The construct according to clause 1, wherein the labile peptide linker comprises a polypeptide sequence of the format:

[0521] [-(G.sub.aS).sub.x-J-(G.sub.bS).sub.y--].sub.z [0522] wherein [0523] a is 1 to 10; [0524] b is 1 to 10; [0525] x is 1 to 10; [0526] y is 1 to 10 [0527] z is 1 to 10 and [0528] J is W, F, Y, L or M; such as W, F or Y. [0529] 12. The construct according to any one of clauses 7 to 11, wherein a is 4, b is 4 and z is 1. [0530] 13. The construct according to any one of clauses 1 to 12, wherein the antigen-binding fragments are selected from the group consisting of: VLs, V-NARs, scFvs, Fab fragments, F(ab')2 fragments or immunoglobulin chain variable domains such as VHHs and VHs. [0531] 14. A pharmaceutical composition comprising the construct according to any one of clauses 1 to 13 for use as a medicament for the treatment of diseases of the GIT administered by oral administration. [0532] 15. A polypeptide comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0533] The present invention will now be further described by means of the following non-limiting examples.

EXAMPLES

Example 1: The Trypsin Protease Assay

[0534] To test the lability of labile peptide linkers, the Trypsin Protease Assay was developed. This assay is performed as follows.

[0535] A buffered (10 mM acetic acid, pH 3.2, containing 0.01% thimerosal) aqueous suspension of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated Trypsin-agarose beads (trypsin from bovine pancreas; T4019; Sigma Aldrich) is used for the assay. The beads are washed 3 times with water (250 .mu.l beads+1.25 ml water) followed by washing 5 times with Trypsin buffer (TRYP buffer; 1 mM Tris-HCl, 20 mM CaCl2 [pH 8.0]). Finally, the resin is resuspended in TRYP buffer as a 50% (v/v) suspension.

[0536] 100 .mu.l of a 2 mg/ml construct solution is mixed with 225 .mu.l 50% (v/v) immobilized TPCK-treated Trypsin in TRYP buffer. After time intervals such as 0, 10, 15, 30, 45 and 60 minutes of incubation at 37.degree. C. in a shaker, samples are taken as follows: resin is pelleted by a 1 min centrifugation step at 500.times.g, and a 40 .mu.l sample is taken from the supernatant and mixed with 2.times. sample loading buffer (such as Laemmli buffer). The remaining suspension is mixed again, and put back at 37.degree. C. in the shaker.

[0537] For analysis, 15 .mu.l of each sample is mixed with 5 .mu.l 4.times. loading dye, boiled for 10 mins and 15 .mu.l is loaded per lane on a polyacrylamide gel (such as NuPAGE 10% acrylamide Bis-Tris gel). Gels are run in SDS-MES buffer at 200 V for 35 mins. Gels are fixed in 40% methanol, 7% acetic acid for 30 mins and stained in colloidal Coomassie Brilliant Blue stain overnight. Gels are destained in water before imaging (such as using ImageQuant LAS4000 with 7 secs exposure). The quantity of intact constructs relative to cleaved constituent polypeptides can be assessed by comparing the corresponding bands in each time point lane.

Example 2: The Chymotrypsin Protease Assay

[0538] The Trypsin Protease Assay protocol can be used wherein Trypsin beads are substituted with Chymotrypsin beads.

Example 3: The Faecal Extract Protease Assay

[0539] To test the lability of labile peptide linkers, the Faecal Extract Protease Assay was developed. Faecal extract is a physiologically relevant matrix in particular for polypeptides targeted to the large intestine. This assay is performed as follows.

[0540] Human faecal samples from multiple individuals are turned into slurries with addition of 1.times.PBS at a ratio of 1, 2 or 3 mLs 1.times.PBS per gram of faeces. The slurries are then pooled (such that one pool represents the combined protease output from the faeces of multiple individuals), centrifuged and the supernatants removed, aliquoted and stored at -70 degrees C. This process removes the faecal matrix, including any cellular material. For digestion, constructs are incubated at a concentration of 160.16 .mu.g/ml at 37.degree. C. in pooled human faecal supernatant for 60 mins in the absence of BSA carrier. Aliquots are taken after time intervals such as 0, 10, 30 and 60 minutes and are mixed 1:1 in Protease Stop Solution (1.times.PBS+2.times. protease inhibitor cocktail (Sigma)+1% PMSF) and immediately frozen at -80.degree. C.

[0541] For analysis, samples are defrosted and 15 .mu.l of each sample is mixed with 5 .mu.l 4.times. loading dye, boiled for 10 mins and 15 .mu.l is loaded per lane on a polyacrylamide gel (such as NuPAGE 10% acrylamide Bis-Tris gel). Gels are run in SDS-MES buffer at 200 V for 35 mins. Gels are fixed in 40% methanol, 7% acetic acid for 30 mins and stained in colloidal Coomassie Brilliant Blue stain overnight. Gels were destained in water before imaging (such as using ImageQuant LAS4000 with 7 secs exposure). The quantity of intact constructs relative to cleaved constituent polypeptides can be assessed by comparing the corresponding higher and lower molecular weight bands in each time point lane.

Example 4: Production of Constructs

[0542] Homobihead constructs containing a range of different linker sequences linking either (a) two anti-TcdA (Clostridium difficile toxin A) ICVDs were expressed in S.cerevisiae and (b) two anti-TNF-alpha ICVDs were designed and expressed in E. coli. The anti-TcdA constructs were homobiheads of ICVD ID1A, separated by different linkers. The anti-TNF-alpha constructs were homobiheads of ICVD ID5F, encoded by polynucleotides which also included Flag-6His tags.

Anti-TcdA Constructs

[0543] Homobihead constructs ID3A, ID4A and ID25A to ID28A were expressed from S. cerevisiae. These constructs do not carry any protein tags at either terminus. ID25A to ID28A, containing the labile peptide linkers, were generated by overlap PCR using ID1A encoding DNA as a template and cloned into the SacI/HindIII sites of shuttle vector pUR4547 (BAC) capable of replication in E. coli and S. cerevisiae. Expression, and full secretion into the supernatant, from S. cerevisiae was achieved from the shuttle vector via galactose induction. Finally, the constructs were purified using Capto S cation exchange resin (GE) and dialysed into 1.times.PBS.

[0544] Expression in S. cerevisiae and subsequent storage was carried out according to the Yeast Expression Protocol as detailed above.

Anti-TNF-Alpha Constructs

[0545] ID55F to ID60F were cloned directly into the SfiI/BsteII sites of vector pMEK222 (QVQ). ID55F to ID60F carry C-terminal Flag-6His tags. The resulting plasmids were transformed into E. coli BL21 DE3 (Novagen) for expression and transport to the periplasm. Following overnight growth in Autoinduction medium Terrific broth (Formedium), cells were pelleted, frozen at -80 degrees C. and re-suspended in 1/10 the original volume of 1.times.PBS to disrupt the outer membrane. ID55F to ID60F were purified from the supernatant by AKTA Prime FPLC using a Ni-NTA column to bind the His tags, followed by imidazole elution and dialysis into 1.times.PBS.

[0546] The sequences of the linkers are given in Table 1 below.

TABLE-US-00002 TABLE 1 Linker Construct Construct SEQ ID SEQ ID Name ICVD Target Linker Sequence Number Number ID3A TcdA GGGGSGGGGSGGGGSGGGGS 1 7 ID25A TcdA GGGGSKGGGGS 2 8 ID26A TcdA GGGGSDKDGGGGS 3 9 ID27A TcdA GGGGSDDDDKGGGGS 4 10 ID28A TcdA KGGGGSGGGGSK 5 11 ID4A TcdA RGGGGSRGGGGSR 6 38 ID55F Human TNF- GGGGSGGGGSGGGGSGGGGS 1 12 alpha ID57F Human TNF- GGGGSKGGGGS 2 13 alpha ID58F Human TNF- GGGGSDKDGGGGS 3 14 alpha ID59F Human TNF- GGGGSDDDDKGGGGS 4 15 alpha ID60F Human TNF- KGGGGSGGGGSK 5 16 alpha ID56F Human TNF- RGGGGSRGGGGSR 6 17 alpha

Example 5: Assaying the Lability of Anti-TcdA Constructs Using the Trypsin Protease Assay

[0547] The lability of the constructs of Example 4 targeting TcdA were assayed using The Trypsin Protease Assay described in Example 1. The results of the assay are shown in SDS-PAGE gels in FIGS. 1 to 3 wherein M=molecular weight marker; lane 1=ICVD before addition of trypsin beads, lane 2=0 min digestion, lane 3=10 min digestion, lane 4=25 min digestion, lane 5=60 min digestion, lane 6=105 min digestion and lane 7=160 min digestion with trypsin beads.

[0548] It can be seen that ID3A remained substantially intact over all time periods tested (FIG. 1). ID25A (FIG. 2) was completely cleaved very quickly after between 0 and 10 minutes of incubation. The shielding D residues in ID26A (FIG. 2) resulted in gradual cleavage of the linker from between 0 to 10 minutes to complete cleavage after 105 minutes. The labile residues situated at either terminus of the ID28A linker (FIG. 3) and the additional N-side shielding D residues in ID27A (FIG. 3) both resulted in slower cleavage, still ongoing after 160 minutes of incubation. The constructs in order of least to most labile were ID3A>ID28A>ID27A>ID26A>ID25A.

[0549] Uncleaved constructs running at molecular weights of approximately 35 kDa are visible in lanes corresponding to early time points (FIGS. 2 and 3). The cleaved constructs (i.e. constituent monomer ICVDs) are visible in lanes corresponding to later time points running at molecular weights of approximately 18 kDa. It is clear therefore from visual inspection of these gels that greater than 50% by mass of constructs ID25A, ID26A, ID27A and ID28A was cleaved into first and second immunoglobulin chain variable domains after 160 minutes (or earlier in some instances) after mixing in the Trypsin Protease Assay (lane 7). These are constructs of the invention comprising labile peptide linkers.

[0550] It is also clear from visual inspection of the gel in FIG. 1 that the only significant bands visible are those corresponding to the intact ID3A construct (running at a molecular weight of approximately 37 kDa). Therefore, ID3A was not cleaved into first and second immunoglobulin chain variable domains at any time point including after 160 minutes (lane 7) after mixing in the Trypsin Protease Assay. ID3A does not comprise a labile peptide linker and is not a construct according to the invention.

[0551] Furthermore, it is clear from visual inspection of the gels in FIGS. 2 and 3 that the first immunoglobulin chain variable domain and the second immunoglobulin chain variable domain in these constructs are themselves substantially resistant to one or more proteases present in the intestinal tract. It can be seen that there are no significant bands visible which correspond to fragments of ICVD monomers (the intact monomers running at a molecular weight of approximately 18 kDa) and thus at least 70% by mass of the immunoglobulin chain variable domains have clearly remained uncleaved after all time points tested including 160 minutes after mixing in the Trypsin Protease Assay.

Example 6: Assaying the Lability of Anti-TcdA and Anti-TNF-Alpha Constructs Using the Faecal Protease Assay

[0552] The lability of the constructs of Example 4 targeting TcdA and targeting TNF-alpha were assayed using The Faecal Protease Assay described in Example 3. The results of the assay are shown in SDS-PAGE gels in FIG. 4 (anti-TcdA constructs) and FIG. 5 (anti-TNF-alpha constructs). Lanes are labelled according to the construct tested and the period of incubation in minutes. `X` indicates lane containing no ICVD (only faecal extract). In FIG. 5, `B` indicates bihead and `M` indicates cleaved monomers.

[0553] Anti-TcdA Constructs

[0554] It can be seen from FIG. 4 that ID3A remained substantially intact over all time periods tested. ID25A was completely cleaved very quickly after between 0 and 10 minutes of incubation. The shielding D residues in ID26A, the additional N-side shielding D residues in ID27A and the labile residues situated at either terminus of the ID28A linker all resulted in slower cleavage relative to ID25A. Digestion of ID26A, ID27A and ID28A was still ongoing after 30 minutes of incubation.

Anti-TNF-Alpha Constructs

[0555] It can be seen from FIG. 5 that ID55F remained substantially intact over all time periods tested. ID56F underwent instant total cleavage. ID57F was completely cleaved very quickly after between 0 and 10 minutes of incubation. The shielding D residues in ID58F, the labile residues situated at either terminus of the ID60F linker and the additional N-side shielding D restudies in ID59F all resulted in slower cleavage relative to ID57F. Digestion of ID58F, ID59F and ID60F was still ongoing after 60 minutes of incubation.

Faecal Protease Assay Summary

[0556] It is apparent that lysine residues in the central region (but not peripheral regions) of the labile peptide linker increase lability to faecal proteases. Lability is reduced by shielding the lysine residues using flanking aspartate residues. The lability of the tested constructs in the Faecal Protease Assay is summarised in Tables 2 and 3 below.

TABLE-US-00003 TABLE 2 Anti-TcdA Linker SEQ ID Construct Name Linker Sequence Lability Number ID3A GGGGSGGGGSGGGGSGGGGS Substantially no 1 cleavage ID28A KGGGGSGGGGSK Minor cleavage 5 ID26A GGGGSDKDGGGGS Some cleavage 3 ID27A GGGGSDDDDKGGGGS Substantial 4 cleavage ID25A GGGGSKGGGGS Total cleavage 2

TABLE-US-00004 TABLE 3 Anti-TNF-alpha Linker SEQ ID Construct Name Linker Sequence Lability Number ID55F GGGGSGGGGSGGGGSGGGGS Substantially no 1 cleavage ID59F GGGGSDDDDKGGGGS Minimal cleavage 4 ID60F KGGGGSGGGGSK Minor cleavage 5 ID58F GGGGSDKDGGGGS Some cleavage 3 ID57F GGGGSKGGGGS Total cleavage 2 ID56F RGGGGSRGGGGSR Instant cleavage 6

Example 7: Stability of Anti-TcdA Constructs During Storage

[0557] The anti-TcdA constructs (ID3A, ID25A, ID25A, ID27A and ID28A) were tested for their stability over time during storage. Samples were taken after 0, 2, 4 or 9 days of refrigeration at 4.degree. C. (FIGS. 6 and 7). It can be seen that the SDS-PAGE gels contain bands corresponding to uncleaved bihead constructs and do not contain bands for cleaved monomers. Accordingly, the constructs are stable for at least 9 days under these conditions.

[0558] These uncleaved constructs ran with a molecular weight of approximately 35 kDa and constituent monomers would be expected to have an approximate molecular weight of 18 kDa. It is clear from visual inspection of the gels in FIGS. 6 and 7 that no significant bands are visible at a lower molecular weight than the 35 kDa bands corresponding to the intact constructs themselves and thus clearly no more than 10% by mass of these constructs is cleaved into first and second immunoglobulin chain variable domains after producing the constructs using the Yeast Expression Protocol (with 0 days storage) and also after a storage period of 2, 4 or 9 days.

[0559] Furthermore, it is clear that the constituent immunoglobulin chain variable domains are substantially resistant to yeast proteases because no significant bands running at a lower molecular weight than that of a single ICVD monomer (running at approximately 18 kDa) are visible in FIG. 6 or 7. Accordingly, it is clear that no more than 10% by mass of the first or second immunoglobulin chain variable domain is cleaved after producing the first or second immunoglobulin chain variable domain using the Yeast Expression Protocol (with 0 days storage) and also after a storage period of 2, 4 or 9 days.

Example 8: Production of Constructs Incorporating Chymotrypsin-Labile Linkers

[0560] Homobihead or heterobihead constructs containing a range of different linker sequences containing chymotrypsin-labile sites (W, F, Y, L or M; particularly W, F or Y) may be designed and expressed in a suitable host such as S. cerevisiae, using the methods analogous to those detailed in Example 4 above. A suitable construct is provided in Table 4.

TABLE-US-00005 TABLE 4 Linker SEQ ID ICVD Target Linker Sequence Number TcdA GGGGSYGGGGS 37

[0561] The lability of such constructs is then assayed using the Chymotrypsin Protease Assay and the Faecal Protease Assay.

Example 9: Testing the Stability of ID4A During Production and Storage

[0562] Anti-TcdA construct ID4A was expressed in S.cerevisiae using The Yeast Expression Protocol. ID4A is a homobihead of ICVD ID1A and does not carry any protein tag at either terminus. The sequence of the linker is given in Table 1 above.

[0563] ID4A was tested for its stability over time during production and storage. Samples were taken after 0, 2, 4 or 9 days of refrigeration at 4.degree. C. (FIG. 8). It can be seen that every lane of the SDS-PAGE gel contains bands corresponding to cleaved monomers, all running at an approximate molecular weight of 10 kDa and no lane clearly contains a band corresponding to uncleaved bihead. It appears that the linker used in ID4A was cleaved by yeast proteases during production.

[0564] It is therefore clear from visual inspection of the gel in FIG. 8 that more than 10% (in fact, 100%) by mass of ID4A is cleaved into first and second immunoglobulin chain variable domains after producing the construct using the Yeast Expression Protocol (with 0 days storage). Constructs of the invention are stable to yeast proteases and therefore ID4A is not a construct of the invention.

[0565] Throughout the specification and the claims which follow, unless the context requires otherwise, the word `comprise`, and variations such as `comprises` and `comprising`, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps. All patents and patent applications mentioned throughout the specification of the present invention are herein incorporated in their entirety by reference. The invention embraces all combinations of preferred and more preferred groups and suitable and more suitable groups and embodiments of groups recited above.

Sequence CWU 1

1

5515PRTArtificial SequenceB10F1arm CDR1 1Ser Tyr Tyr Met Gly 1 5 217PRTArtificial SequenceB10F1arm CDR2 2Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val Lys 1 5 10 15 Gly 313PRTArtificial SequenceB10F1arm CDR3 3Ser Leu Thr Tyr Tyr Gly His Ser Ala His Tyr Asp Tyr 1 5 10 45PRTArtificial SequenceQ31B1arm CDR1 4Ser Tyr Thr Met Gly 1 5 517PRTArtificial SequenceQ31B1arm CDR2 5Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val Lys 1 5 10 15 Gly 615PRTArtificial SequenceQ31B1arm CDR3 6His Thr Thr Ser Gly Val Pro Val His Glu Arg Ser Tyr Ala Tyr 1 5 10 15 75PRTArtificial SequenceQ35H8 CDR1 7Ser Phe Thr Met Gly 1 5 817PRTArtificial SequenceQ35H8 CDR2 8Gly Lys Ser Arg Asp Gly Arg Thr Thr Tyr Tyr Ser Asn Ser Val Lys 1 5 10 15 Gly 915PRTArtificial SequenceQ35H8 CDR3 9His Thr Thr Ser Gly Val Pro Val Arg Val Lys Ser Tyr Ala Tyr 1 5 10 15 10122PRTArtificial SequenceB10F1arm 10Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Phe Ser Ser Tyr 20 25 30 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Leu Thr Tyr Tyr Gly His Ser Ala His Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 11124PRTArtificial SequenceQ31B1arm 11Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val His Glu Arg Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 12124PRTArtificial SequenceQ35H8 12Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Pro Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Pro Leu Ser Ser Phe 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Leu 35 40 45 Gly Gly Lys Ser Arg Asp Gly Arg Thr Thr Tyr Tyr Ser Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Asp Arg Asp Asp Ala Gln Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Asn Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val Arg Val Lys Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 13122PRTArtificial SequenceID1B 13Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Phe Ser Ser Tyr 20 25 30 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Leu Thr Tyr Tyr Gly Arg Ser Ala Arg Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 14124PRTArtificial SequenceID2B 14Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val Arg Glu Arg Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 15124PRTArtificial SequenceID3B 15Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Trp Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ala Pro Leu Ser Ser Phe 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Leu 35 40 45 Gly Gly Lys Ser Arg Asp Gly Arg Thr Thr Tyr Tyr Ser Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Asp Arg Asp Asp Ala Gln Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Asn Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val Arg Val Lys Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 16266PRTArtificial SequenceID11B 16Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Leu Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val Arg Glu Arg Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 145 150 155 160 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 165 170 175 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 180 185 190 Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val 195 200 205 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 210 215 220 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 225 230 235 240 Ala Ala Ser Leu Thr Tyr Tyr Gly Arg Ser Ala Arg Tyr Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 260 265 17266PRTArtificial SequenceID12B 17Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Trp Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Pro Leu Ser Ser Phe 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Leu 35 40 45 Gly Gly Lys Ser Arg Asp Gly Arg Thr Thr Tyr Tyr Ser Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Asp Arg Asp Asp Ala Gln Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Asn Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val Arg Val Lys Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 145 150 155 160 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 165 170 175 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 180 185 190 Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val 195 200 205 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 210 215 220 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 225 230 235 240 Ala Ala Ser Leu Thr Tyr Tyr Gly Arg Ser Ala Arg Tyr Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 260 265 18124PRTArtificial SequenceID20B 18Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu Ser Ser Tyr 20 25 30 Thr Ile Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser His Asp Gly His Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val Arg Glu Arg Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 19124PRTArtificial SequenceID21B 19Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu Ser Ser Tyr 20 25 30 Thr Ile Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val His Glu Arg Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 20124PRTArtificial SequenceID22B 20Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu Ser Ser Tyr 20 25 30 Thr Ile Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val Arg Glu His Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 21122PRTArtificial SequenceID24B 21Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Phe Ser Ser Tyr 20 25 30 Tyr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Asn Gly Ser Gly Gly Asn His Ile Ser Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Leu Thr Tyr Tyr Gly Arg Ser Ala Arg Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 22122PRTArtificial SequenceID25B 22Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Phe Ser Ser Tyr 20 25 30 Tyr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Leu Thr Tyr Tyr Gly Arg Ser Ala His Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 23122PRTArtificial SequenceID27B 23Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Phe Ser Ser Tyr 20 25 30 Tyr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Leu Thr Tyr Tyr Gly His Ser Ala Arg Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 24266PRTArtificial SequenceID41B 24Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50

55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val His Glu Arg Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 145 150 155 160 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Phe Ser Ser Tyr 165 170 175 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 180 185 190 Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val 195 200 205 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 210 215 220 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 225 230 235 240 Ala Ala Ser Leu Thr Tyr Tyr Gly His Ser Ala Arg Tyr Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 260 265 25266PRTArtificial SequenceID43B 25Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val His Glu Arg Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 145 150 155 160 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Phe Ser Ser Tyr 165 170 175 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 180 185 190 Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val 195 200 205 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 210 215 220 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 225 230 235 240 Ala Ala Ser Leu Thr Tyr Tyr Gly His Ser Ala His Tyr Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 260 265 26124PRTArtificial SequenceID45B 26Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val Arg Glu Arg Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 27124PRTArtificial SequenceID46B 27Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val His Glu Arg Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 28124PRTArtificial SequenceID49B 28Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val Phe Glu Arg Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 29122PRTArtificial SequenceB10F1 29Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Leu Thr Tyr Tyr Gly Arg Ser Ala Arg Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 30124PRTArtificial SequenceQ31B1 30Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Leu Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val Arg Glu Arg Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 3128DNAArtificial Sequence3' primer 31tcttaactag tgaggagacg gtgacctg 283217DNAArtificial SequenceM13.rev 32caggaaacag ctatgac 173316DNAArtificial SequenceM13.fw 33gtaaaacgac ggccag 16341100DNAArtificial SequencePolynucleotide sequence encoding ID11B 34gagctcatca cacaaacaaa caaaacaaaa tgatgagatt tccttcaatt tttactgccg 60ttttattcgc agcatcctcc gcattagctg ctccagtcaa cactacaaca gaagatgaaa 120cggcacaaat tccggctgaa gctgtcatcg gttactcaga tttagaaggg gatttcgatg 180ttgctgtttt gccattttcc aacagcacaa ataacgggtt attgtttata aatactacta 240ttgccagcat tgctgctaaa gaagaagggg tatctctcga gaaaagagat gtgcagctgc 300aggagtctgg gggaggattg gtgcaggctg gggactctct gagactctcc tgtgcagcct 360ctggacgtac cttaagtagc tataccatgg gctggttccg tcaggcgcca gagaaggagc 420gcgagtttgt agcaggtagt agccgggatg gtcgtacaaa ctactatgca aactccgtga 480agggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg caaatgaaca 540gcctgaaacc tgaggacacg gccgtttatt actgcgcagc gcacactacc agcggtgtcc 600cggtccggga gagatcgtat gcctactggg gccaggggac ccaggtcact gtctcctcag 660gtggaggcgg ttcaggcgga ggtggctctg gcggtggcgg aagtggtgga ggcggttcag 720atgtgcagct gcaagagagc gggggaggat tggtgcaggc tgggggctct ctgagactct 780cctgtgcagc ctctggacgt accttcagta gctattacat gggctggttc cgccaggctc 840cagggaagga acgtgagttt gtagcagcta ttaacgggag tggtggtaac agaatctctg 900ctgactccgt gaagggccga ttcaccatct ccagagacaa cgccaagaac acggtgtatc 960tgcaactgaa cagcctgaaa cctgaggaca cggccgttta ttactgtgca gcctccctga 1020cttactatgg tcgttcggca cgttatgact actggggcca ggggacccag gtcaccgtct 1080cctcataatg acttaagctt 1100351100DNAArtificial SequencePolynucleotide sequence encoding ID12B 35gagctcatca cacaaacaaa caaaacaaaa tgatgagatt tccttcaatt tttactgccg 60ttttattcgc agcatcctcc gcattagctg ctccagtcaa cactacaaca gaagatgaaa 120cggcacaaat tccggctgaa gctgtcatcg gttactcaga tttagaaggg gatttcgatg 180ttgctgtttt gccattttcc aacagcacaa ataacgggtt attgtttata aatactacta 240ttgccagcat tgctgctaaa gaagaagggg tatctctcga gaaaagagat gtgcagctgc 300aggagtctgg gggagggtgg gtgcaggctg gggactctct gagactctcc tgtgtagctt 360ctgggagacc cttaagtagc tttaccatgg gctggttccg tcaggcgcca gagaaggagc 420gcgagtttct aggaggtaag agccgggatg gccgtacgac atactattcg aactccgtga 480agggccgatt caccatcgac agagacgacg cccagaacac ggtgtatctg caaatgaaca 540gcctgaatcc tgacgacacg gccgtttatt actgcgcagc gcacactacc agcggtgtcc 600cggtccgggt gaaatcgtat gcctactggg gccaggggac ccaggtcact gtctcctcag 660gtggaggcgg ttcaggcgga ggtggctctg gcggtggcgg aagtggtgga ggcggttcag 720atgtgcagct gcaagagagc gggggaggat tggtgcaggc tgggggctct ctgagactct 780cctgtgcagc ctctggacgt accttcagta gctattacat gggctggttc cgccaggctc 840cagggaagga acgtgagttt gtagcagcta ttaacgggag tggtggtaac agaatctctg 900ctgactccgt gaagggccga ttcaccatct ccagagacaa cgccaagaac acggtgtatc 960tgcaactgaa cagcctgaaa cctgaggaca cggccgttta ttactgtgca gcctccctga 1020cttactatgg tcgttcggca cgttatgact actggggcca ggggacccag gtcaccgtct 1080cctcataatg acttaagctt 1100361180DNAArtificial SequencePolynucleotide sequence encoding ID41B 36cactataggg cgaattgaag gaaggccgtc aaggccgcat gagctcatca cacaaacaaa 60caaaacaaaa tgatgagatt tccttcaatt tttactgccg ttttattcgc agcatcctcc 120gcattagctg ctccagtcaa cactacaaca gaagatgaaa cggcacaaat tccggctgaa 180gctgtcatcg gttactcaga tttagaaggg gatttcgatg ttgctgtttt gccattttcc 240aacagcacaa ataacgggtt attgtttata aatactacta ttgccagcat tgctgctaaa 300gaagaagggg tatctctcga gaaaagagat gtgcagctgc aggagtctgg gggaggattg 360gtgcaggctg gggactctct gagactctcc tgtgcagcct ctggagctac cttaagtagc 420tataccatgg gctggttccg tcaggcgcca gagaaggagc gcgagtttgt agcaggtagt 480agccgggatg gtcgtacaaa ctactatgca aactccgtga agggccgatt caccatctcc 540agagacaacg ccaagaacac ggtgtatctg caaatgaaca gcctgaaacc tgaggacacg 600gccgtttatt actgcgcagc gcacactacc agcggtgtcc cggtccatga gagatcgtat 660gcctactggg gccaggggac ccaggtcact gtctcctcag gtggaggcgg ttcaggcgga 720ggtggctctg gcggtggcgg aagtggtgga ggcggttcag atgtgcagct gcaagagagc 780gggggaggat tggtgcaggc tgggggctct ctgagactct cctgtgcagc ctctggagct 840accttcagta gctattacat gggctggttc cgccaggctc cagggaagga acgtgagttt 900gtagcagcta ttaacgggag tggtggtaac agaatctctg ctgactccgt gaagggccga 960ttcaccatct ccagagacaa cgccaagaac acggtgtatc tgcaactgaa cagcctgaaa 1020cctgaggaca cggccgttta ttactgtgca gcctccctga cttactatgg tcattcggca 1080cgttatgact actggggcca ggggacccag gtcaccgtct cctcataatg acttaagctt 1140ctgggcctca tgggccttcc tttcactgcc cgctttccag 1180371180DNAArtificial SequencePolynucleotide sequence encoding ID43B 37cactataggg cgaattgaag gaaggccgtc aaggccgcat gagctcatca cacaaacaaa 60caaaacaaaa tgatgagatt tccttcaatt tttactgccg ttttattcgc agcatcctcc 120gcattagctg ctccagtcaa cactacaaca gaagatgaaa cggcacaaat tccggctgaa 180gctgtcatcg gttactcaga tttagaaggg gatttcgatg ttgctgtttt gccattttcc 240aacagcacaa ataacgggtt attgtttata aatactacta ttgccagcat tgctgctaaa 300gaagaagggg tatctctcga gaaaagagat gtgcagctgc aggagtctgg gggaggattg 360gtgcaggctg gggactctct gagactctcc tgtgcagcct ctggagctac cttaagtagc 420tataccatgg gctggttccg tcaggcgcca gagaaggagc gcgagtttgt agcaggtagt 480agccgggatg gtcgtacaaa ctactatgca aactccgtga agggccgatt caccatctcc 540agagacaacg ccaagaacac ggtgtatctg caaatgaaca gcctgaaacc tgaggacacg 600gccgtttatt actgcgcagc gcacactacc agcggtgtcc cggtccatga gagatcgtat 660gcctactggg gccaggggac ccaggtcact gtctcctcag gtggaggcgg ttcaggcgga 720ggtggctctg gcggtggcgg aagtggtgga ggcggttcag atgtgcagct gcaagagagc 780gggggaggat tggtgcaggc tgggggctct ctgagactct cctgtgcagc ctctggagct 840accttcagta gctattacat gggctggttc cgccaggctc cagggaagga acgtgagttt 900gtagcagcta ttaacgggag tggtggtaac agaatctctg ctgactccgt gaagggccga 960ttcaccatct ccagagacaa cgccaagaac acggtgtatc tgcaactgaa cagcctgaaa 1020cctgaggaca cggccgttta ttactgtgca gcctccctga cttactatgg tcattcggca 1080cattatgact actggggcca ggggacccag gtcaccgtct cctcataatg acttaagctt 1140ctgggcctca tgggccttcc tttcactgcc cgctttccag 118038372DNAArtificial SequencePolynucleotide sequence encoding B10F1arm 38gatgtgcagc tgcaagagag cgggggagga ttggtgcagg ctgggggctc tctgagactc 60tcctgtgcag cctctggagc taccttcagt agctattaca tgggctggtt ccgccaggct 120ccagggaagg aacgtgagtt tgtagcagct attaacggga gtggtggtaa cagaatctct 180gctgactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240ctgcaactga acagcctgaa acctgaggac acggccgttt attactgtgc agcctccctg 300acttactatg gtcattcggc acattatgac tactggggcc aggggaccca ggtcaccgtc 360tcctcataat ga 37239378DNAArtificial SequencePolynucleotide sequence encoding Q31B1arm 39gatgtgcagc tgcaggagtc tgggggagga ttggtgcagg ctggggactc tctgagactc 60tcctgtgcag cctctggagc taccttaagt agctatacca tgggctggtt ccgtcaggcg 120ccagagaagg agcgcgagtt tgtagcaggt agtagccggg atggtcgtac aaactactat 180gcaaactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgcgc agcgcacact 300accagcggtg tcccggtcca tgagagatcg tatgcctact ggggccaggg gacccaggtc 360actgtctcct cataatga 37840378DNAArtificial SequencePolynucleotide sequence encoding Q35H8arm 40gatgtgcagc tgcaggagtc tgggggaggg tgggtgcagg ctggggactc tctgagactc 60tcctgtgtag cttctgggag acccttaagt agctttacca tgggctggtt ccgtcaggcg 120ccagagaagg agcgcgagtt tctaggaggt aagagccggg atggccgtac gacatactat 180tcgaactccg tgaagggccg attcaccatc gacagagacg acgcccagaa cacggtgtat 240ctgcaaatga acagcctgaa tcctgacgac acggccgttt attactgcgc agcgcacact 300accagcggtg tcccggtccg ggtgaaatcg tatgcctact ggggccaggg gacccaggtc 360actgtctcct cataatga 37841543PRTArtificial SequenceID1C 41Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Ser Asp Val Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Ile Asn Arg Ser Gly Ser Asp Ser Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Glu Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Arg Ser Asp Cys Ile Gly Tyr Gly Cys Arg Arg Val Ser 100 105 110 Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala 145 150 155 160 Gly Gly Ser Leu Arg

Leu Ser Cys Val Ile Ser Gly Met Asp Phe Ser 165 170 175 His Lys Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 180 185 190 Phe Val Ala Ser Ile Thr Thr Arg Ala Ser Thr His Tyr Ala Asp Ser 195 200 205 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 210 215 220 Tyr Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 225 230 235 240 Cys Asn Ser Glu Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 245 250 255 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 260 265 270 Gly Gly Gly Gly Ser Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu 275 280 285 Val Gln Ala Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala 290 295 300 Thr Leu Ser Ser Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Glu Lys 305 310 315 320 Glu Arg Glu Phe Val Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr 325 330 335 Tyr Ala Asn Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 340 345 350 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 355 360 365 Ala Val Tyr Tyr Cys Ala Ala His Thr Thr Ser Gly Val Pro Val Arg 370 375 380 Glu Arg Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 385 390 395 400 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 405 410 415 Gly Gly Gly Gly Ser Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu 420 425 430 Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala 435 440 445 Thr Phe Ser Ser Tyr Tyr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys 450 455 460 Glu Arg Glu Phe Val Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile 465 470 475 480 Ser Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 485 490 495 Lys Asn Thr Val Tyr Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr 500 505 510 Ala Val Tyr Tyr Cys Ala Ala Ser Leu Thr Tyr Tyr Gly Arg Ser Ala 515 520 525 Arg Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 530 535 540 42543PRTArtificial SequenceID3C 42Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu Ser Ser Tyr 20 25 30 Thr Ile Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val Arg Glu Arg Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 145 150 155 160 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Ser Asp Val Tyr 165 170 175 Ala Ile Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe Val 180 185 190 Ala Thr Ile Asn Arg Ser Gly Ser Asp Ser Tyr Tyr Ala Asp Ser Val 195 200 205 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 210 215 220 Leu Gln Met Asn Ser Leu Lys Pro Glu Glu Thr Ala Val Tyr Tyr Cys 225 230 235 240 Ala Ala Ser Arg Ser Asp Cys Ile Gly Tyr Gly Cys Arg Arg Val Ser 245 250 255 Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 260 265 270 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala 290 295 300 Gly Gly Ser Leu Arg Leu Ser Cys Val Ile Ser Gly Met Asp Phe Ser 305 310 315 320 His Lys Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 325 330 335 Phe Val Ala Ser Ile Thr Thr Arg Ala Ser Thr His Tyr Ala Asp Ser 340 345 350 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 355 360 365 Tyr Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 370 375 380 Cys Asn Ser Glu Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 385 390 395 400 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 405 410 415 Gly Gly Gly Gly Ser Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu 420 425 430 Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala 435 440 445 Thr Phe Ser Ser Tyr Tyr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys 450 455 460 Glu Arg Glu Phe Val Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile 465 470 475 480 Ser Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 485 490 495 Lys Asn Thr Val Tyr Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr 500 505 510 Ala Val Tyr Tyr Cys Ala Ala Ser Leu Thr Tyr Tyr Gly Arg Ser Ala 515 520 525 Arg Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 530 535 540 43543PRTArtificial SequenceID5C 43Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val His Glu Arg Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 145 150 155 160 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Phe Ser Ser Tyr 165 170 175 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 180 185 190 Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val 195 200 205 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 210 215 220 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 225 230 235 240 Ala Ala Ser Leu Thr Tyr Tyr Gly His Ser Ala Arg Tyr Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 260 265 270 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val 275 280 285 Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu 290 295 300 Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Ser Asp Val Tyr Ala Met 305 310 315 320 Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe Val Ala Thr 325 330 335 Ile Asn Arg Ser Gly Ser Asp Ser Tyr Tyr Ala Asp Ser Val Lys Gly 340 345 350 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 355 360 365 Met Asn Ser Leu Lys Pro Glu Glu Thr Ala Val Tyr Tyr Cys Ala Ala 370 375 380 Ser Arg Ser Asp Cys Ile Gly Tyr Gly Cys Arg Arg Val Ser Gln Asp 385 390 395 400 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly 405 410 415 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 420 425 430 Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 435 440 445 Ser Leu Arg Leu Ser Cys Val Ile Ser Gly Met Asp Phe Ser His Lys 450 455 460 Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 465 470 475 480 Ala Ser Ile Thr Thr Arg Ala Ser Thr His Tyr Ala Asp Ser Val Lys 485 490 495 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 500 505 510 Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 515 520 525 Ser Glu Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 530 535 540 44543PRTArtificial SequenceID6C 44Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Ser Asp Val Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Ile Asn Arg Ser Gly Ser Asp Ser Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Glu Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Arg Ser Asp Cys Ile Gly Tyr Gly Cys Arg Arg Val Ser 100 105 110 Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala 145 150 155 160 Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu Ser 165 170 175 Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu 180 185 190 Phe Val Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn 195 200 205 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 210 215 220 Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 225 230 235 240 Tyr Cys Ala Ala His Thr Thr Ser Gly Val Pro Val His Glu Arg Ser 245 250 255 Tyr Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 260 265 270 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala 290 295 300 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Phe Ser 305 310 315 320 Ser Tyr Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 325 330 335 Phe Val Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp 340 345 350 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 355 360 365 Val Tyr Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 370 375 380 Tyr Cys Ala Ala Ser Leu Thr Tyr Tyr Gly His Ser Ala Arg Tyr Asp 385 390 395 400 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly 405 410 415 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 420 425 430 Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 435 440 445 Ser Leu Arg Leu Ser Cys Val Ile Ser Gly Met Asp Phe Ser His Lys 450 455 460 Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 465 470 475 480 Ala Ser Ile Thr Thr Arg Ala Ser Thr His Tyr Ala Asp Ser Val Lys 485 490 495 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 500 505 510 Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 515 520 525 Ser Glu Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 530 535 540 45543PRTArtificial SequenceID7C 45Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ile Ser Gly Met Asp Phe Ser His Lys 20 25 30 Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ser Ile Thr Thr Arg Ala Ser Thr His Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ser Glu Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Ala Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu 145 150 155 160 Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg 165 170 175 Glu Phe Val Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala 180 185 190 Asn Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 195 200 205 Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Ala His Thr Thr Ser Gly Val Pro Val His Glu Arg 225 230 235 240 Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly 245 250 255 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 260 265 270 Gly Gly Ser Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln

275 280 285 Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Phe 290 295 300 Ser Ser Tyr Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 305 310 315 320 Glu Phe Val Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala 325 330 335 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 340 345 350 Thr Val Tyr Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val 355 360 365 Tyr Tyr Cys Ala Ala Ser Leu Thr Tyr Tyr Gly His Ser Ala Arg Tyr 370 375 380 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly 385 390 395 400 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 405 410 415 Ser Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly 420 425 430 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Ser Asp Val 435 440 445 Tyr Ala Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe 450 455 460 Val Ala Thr Ile Asn Arg Ser Gly Ser Asp Ser Tyr Tyr Ala Asp Ser 465 470 475 480 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 485 490 495 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Glu Thr Ala Val Tyr Tyr 500 505 510 Cys Ala Ala Ser Arg Ser Asp Cys Ile Gly Tyr Gly Cys Arg Arg Val 515 520 525 Ser Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 530 535 540 46543PRTArtificial SequenceID8C 46Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val His Glu Arg Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 145 150 155 160 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Phe Ser Ser Tyr 165 170 175 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 180 185 190 Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val 195 200 205 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 210 215 220 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 225 230 235 240 Ala Ala Ser Leu Thr Tyr Tyr Gly His Ser Ala His Tyr Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 260 265 270 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val 275 280 285 Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu 290 295 300 Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Ser Asp Val Tyr Ala Met 305 310 315 320 Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe Val Ala Thr 325 330 335 Ile Asn Arg Ser Gly Ser Asp Ser Tyr Tyr Ala Asp Ser Val Lys Gly 340 345 350 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 355 360 365 Met Asn Ser Leu Lys Pro Glu Glu Thr Ala Val Tyr Tyr Cys Ala Ala 370 375 380 Ser His Ser Asp Cys Ile Gly Tyr Gly Cys His His Val Ser Gln Asp 385 390 395 400 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly 405 410 415 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 420 425 430 Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 435 440 445 Ser Leu Arg Leu Ser Cys Val Ile Ser Gly Met Asp Phe Ser His Lys 450 455 460 Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 465 470 475 480 Ala Ser Ile Thr Thr Arg Ala Ser Thr His Tyr Ala Asp Ser Val Lys 485 490 495 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 500 505 510 Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 515 520 525 Ser Glu Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 530 535 540 47543PRTArtificial SequenceID11C 47Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ile Ser Gly Met Asp Phe Ser His Lys 20 25 30 Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ser Ile Thr Thr Arg Ala Ser Thr His Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ser Glu Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Ala Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu 145 150 155 160 Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg 165 170 175 Glu Phe Val Ala Gly Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala 180 185 190 Asn Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 195 200 205 Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Ala His Thr Thr Ser Gly Val Pro Val His Glu Arg 225 230 235 240 Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly 245 250 255 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 260 265 270 Gly Gly Ser Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln 275 280 285 Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Phe 290 295 300 Ser Ser Tyr Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 305 310 315 320 Glu Phe Val Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala 325 330 335 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 340 345 350 Thr Val Tyr Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val 355 360 365 Tyr Tyr Cys Ala Ala Ser Leu Thr Tyr Tyr Gly His Ser Ala His Tyr 370 375 380 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly 385 390 395 400 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 405 410 415 Ser Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly 420 425 430 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Ser Asp Val 435 440 445 Tyr Ala Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe 450 455 460 Val Ala Thr Ile Asn Arg Ser Gly Ser Asp Ser Tyr Tyr Ala Asp Ser 465 470 475 480 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 485 490 495 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Glu Thr Ala Val Tyr Tyr 500 505 510 Cys Ala Ala Ser His Ser Asp Cys Ile Gly Tyr Gly Cys His His Val 515 520 525 Ser Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 530 535 540 48111PRTArtificial SequenceQ34A3 48Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ile Ser Gly Met Asp Phe Ser His Lys 20 25 30 Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ser Ile Thr Thr Arg Ala Ser Thr His Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Ser Cys Asn 85 90 95 Ser Glu Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 100 105 110 49126PRTArtificial SequenceB4F10 49Gln Val Gln Leu Gln Glu Thr Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Asp Val Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Ile Asn Arg Ser Gly Ser Asp Ser Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Glu Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Arg Ser Asp Cys Ile Gly Tyr Gly Cys Arg Arg Val Ser 100 105 110 Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 50257PRTArtificial SequenceID33A 50Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Ser Asp Val Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Ile Asn Arg Ser Gly Ser Asp Ser Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Glu Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser His Ser Asp Cys Ile Gly Tyr Gly Cys His His Val Ser 100 105 110 Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala 145 150 155 160 Gly Gly Ser Leu Arg Leu Ser Cys Val Ile Ser Gly Met Asp Phe Ser 165 170 175 His Lys Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 180 185 190 Phe Val Ala Ser Ile Thr Thr Arg Ala Ser Thr His Tyr Ala Asp Ser 195 200 205 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 210 215 220 Tyr Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 225 230 235 240 Cys Asn Ser Glu Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 245 250 255 Ser 51257PRTArtificial SequenceID17A 51Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Ser Asp Val Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Ile Asn Arg Ser Gly Ser Asp Ser Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Glu Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Arg Ser Asp Cys Ile Gly Tyr Gly Cys Arg Arg Val Ser 100 105 110 Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala 145 150 155 160 Gly Gly Ser Leu Arg Leu Ser Cys Val Ile Ser Gly Met Asp Phe Ser 165 170 175 His Lys Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 180 185 190 Phe Val Ala Ser Ile Thr Thr Arg Ala Ser Thr His Tyr Ala Asp Ser 195 200 205 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 210 215 220 Tyr Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 225 230 235 240 Cys Asn Ser Glu Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 245 250 255 Ser 522366PRTArtificial Sequence087 TcdB 52Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg 1 5 10 15 Phe Arg Thr Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu 20 25 30 Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys 35 40 45 Leu Lys Asp Ile Asn Ser Leu Thr Asp Ile Tyr Ile Asp Thr Tyr Lys 50 55 60 Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val 65 70 75 80 Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys 85 90 95 Asn Leu His Phe Val Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile 100 105 110 Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn 115 120 125 Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr 130 135 140 Val Val Glu Ser Ala Ile Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn 145 150 155 160 Leu Asn Asp Pro Arg Phe Asp Tyr Asn Lys Phe Phe Arg Lys Arg Met 165

170 175 Glu Ile Ile Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Ala 180 185 190 Gln Arg Glu Glu Asn Pro Glu Leu Ile Ile Asp Asp Ile Val Lys Thr 195 200 205 Tyr Leu Ser Asn Glu Tyr Ser Lys Glu Ile Asp Glu Leu Asn Thr Tyr 210 215 220 Ile Glu Glu Ser Leu Asn Lys Ile Thr Gln Asn Ser Gly Asn Asp Val 225 230 235 240 Arg Asn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Asn Leu Tyr Glu 245 250 255 Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu 260 265 270 Arg Ile Ser Ala Leu Lys Glu Ile Gly Gly Met Tyr Leu Asp Val Asp 275 280 285 Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro 290 295 300 Ser Ser Val Thr Val Asp Phe Trp Glu Met Thr Lys Leu Glu Ala Ile 305 310 315 320 Met Lys Tyr Lys Glu Tyr Ile Pro Glu Tyr Thr Ser Glu His Phe Asp 325 330 335 Met Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala Ser 340 345 350 Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Met Glu Ala 355 360 365 Ser Pro Leu Glu Val Lys Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn 370 375 380 Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val 385 390 395 400 Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro 405 410 415 Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe Ile 420 425 430 Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met 435 440 445 Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr 450 455 460 Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp 465 470 475 480 Leu Leu Met Phe Lys Glu Gly Ser Met Asn Ile His Leu Ile Glu Ala 485 490 495 Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr 500 505 510 Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys 515 520 525 Ala Gln Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ser Leu Gly 530 535 540 Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Ile Val Val Asp Lys Glu 545 550 555 560 Tyr Leu Leu Glu Lys Ile Ser Ser Leu Ala Arg Ser Ser Glu Arg Gly 565 570 575 Tyr Ile His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr Glu 580 585 590 Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Val Leu Phe 595 600 605 Gln Lys Asn Ile Glu Asp Ser Glu Ile Ala Tyr Tyr Tyr Asn Pro Gly 610 615 620 Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Lys Ile Pro Ser Ile Ile 625 630 635 640 Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp 645 650 655 Glu Phe Asn Thr Asp Ile Phe Ala Gly Phe Asp Val Asp Ser Leu Ser 660 665 670 Thr Glu Ile Glu Ala Ala Ile Asp Leu Ala Lys Glu Asp Ile Ser Pro 675 680 685 Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser 690 695 700 Ile Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys 705 710 715 720 Asp Lys Ile Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile Ile 725 730 735 Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg 740 745 750 Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile 755 760 765 Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys Glu 770 775 780 Asn Lys Ile Thr Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu 785 790 795 800 Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp Ile Glu Leu Glu 805 810 815 Glu Lys Val Met Leu Thr Glu Cys Glu Ile Asn Val Ile Ser Asn Ile 820 825 830 Asp Thr Gln Ile Val Glu Glu Arg Ile Glu Glu Ala Lys Asn Leu Thr 835 840 845 Ser Asp Ser Ile Asn Tyr Ile Lys Asp Glu Phe Lys Leu Ile Glu Ser 850 855 860 Ile Ser Asp Ala Leu Cys Asp Leu Lys Gln Gln Asn Glu Leu Glu Asp 865 870 875 880 Ser His Phe Ile Ser Phe Glu Asp Ile Ser Glu Thr Asp Glu Gly Phe 885 890 895 Ser Ile Arg Phe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val Glu 900 905 910 Thr Glu Lys Thr Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu Glu 915 920 925 Ile Ser Lys Ile Lys Gly Thr Ile Phe Asp Thr Val Asn Gly Lys Leu 930 935 940 Val Lys Lys Val Asn Leu Asp Thr Thr His Glu Val Asn Thr Leu Asn 945 950 955 960 Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr Asn Ser Ser Lys Glu 965 970 975 Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln 980 985 990 Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val Val 995 1000 1005 Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro Thr 1010 1015 1020 Leu Ser Glu Gly Leu Pro Ile Ile Ala Thr Ile Ile Asp Gly Val Ser 1025 1030 1035 1040Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser Asp Pro Leu Leu 1045 1050 1055 Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala Val Asn Leu Thr 1060 1065 1070 Thr Ala Thr Thr Ala Ile Ile Thr Ser Ser Leu Gly Ile Ala Ser Gly 1075 1080 1085 Phe Ser Ile Leu Leu Val Pro Leu Ala Gly Ile Ser Ala Gly Ile Pro 1090 1095 1100 Ser Leu Val Asn Asn Glu Leu Val Leu Arg Asp Lys Ala Thr Lys Val 1105 1110 1115 1120Val Asp Tyr Phe Lys His Val Ser Leu Val Glu Thr Glu Gly Val Phe 1125 1130 1135 Thr Leu Leu Asp Asp Lys Ile Met Met Pro Gln Asp Asp Leu Val Ile 1140 1145 1150 Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Val Leu Gly Lys Cys Glu 1155 1160 1165 Ile Trp Arg Met Glu Gly Gly Ser Gly His Thr Val Thr Asp Asp Ile 1170 1175 1180 Asp His Phe Phe Ser Ala Pro Ser Ile Thr Tyr Arg Glu Pro His Leu 1185 1190 1195 1200Ser Ile Tyr Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser 1205 1210 1215 Lys Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp 1220 1225 1230 Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr 1235 1240 1245 Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu Gly Glu Phe Tyr Trp 1250 1255 1260 Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr Leu Lys Pro 1265 1270 1275 1280Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp Ser Asn Thr Arg 1285 1290 1295 Ser Phe Ile Val Pro Ile Ile Thr Thr Glu Tyr Ile Arg Glu Lys Leu 1300 1305 1310 Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu Ser 1315 1320 1325 Gln Tyr Asn Met Gly Ile Asn Ile Glu Leu Ser Glu Ser Asp Val Trp 1330 1335 1340 Ile Ile Asp Val Asp Asn Val Val Arg Asp Val Thr Ile Glu Ser Asp 1345 1350 1355 1360Lys Ile Lys Lys Gly Asp Leu Ile Glu Gly Ile Leu Ser Thr Leu Ser 1365 1370 1375 Ile Glu Glu Asn Lys Ile Ile Leu Asn Ser His Glu Ile Asn Phe Ser 1380 1385 1390 Gly Glu Val Asn Gly Ser Asn Gly Phe Val Ser Leu Thr Phe Ser Ile 1395 1400 1405 Leu Glu Gly Ile Asn Ala Ile Ile Glu Val Asp Leu Leu Ser Lys Ser 1410 1415 1420 Tyr Lys Leu Leu Ile Ser Gly Glu Leu Lys Ile Leu Met Leu Asn Ser 1425 1430 1435 1440Asn His Ile Gln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu 1445 1450 1455 Gln Lys Asn Ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn 1460 1465 1470 Gly Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu 1475 1480 1485 Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser Lys Pro 1490 1495 1500 Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys Val Ile Thr 1505 1510 1515 1520Lys Asp Asn Val Asn Ile Leu Thr Gly Tyr Tyr Leu Lys Asp Asp Ile 1525 1530 1535 Lys Ile Ser Leu Ser Leu Thr Leu Gln Asp Glu Lys Thr Ile Lys Leu 1540 1545 1550 Asn Ser Val His Leu Asp Glu Ser Gly Val Ala Glu Ile Leu Lys Phe 1555 1560 1565 Met Asn Arg Lys Gly Asn Thr Asn Thr Ser Asp Ser Leu Met Ser Phe 1570 1575 1580 Leu Glu Ser Met Asn Ile Lys Ser Ile Phe Val Asn Phe Leu Gln Ser 1585 1590 1595 1600Asn Ile Lys Phe Ile Leu Asp Ala Asn Phe Ile Ile Ser Gly Thr Thr 1605 1610 1615 Ser Ile Gly Gln Phe Glu Phe Ile Cys Asp Glu Asn Asp Asn Ile Gln 1620 1625 1630 Pro Tyr Phe Ile Lys Phe Asn Thr Leu Glu Thr Asn Tyr Thr Leu Tyr 1635 1640 1645 Val Gly Asn Arg Gln Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp 1650 1655 1660 Asp Ser Gly Asp Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr 1665 1670 1675 1680Leu Tyr Gly Ile Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro Asn 1685 1690 1695 Ile Tyr Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Thr Asn Asn 1700 1705 1710 Thr Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu Lys 1715 1720 1725 Ile Asn Val Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser Asn 1730 1735 1740 Asp Gly Asn Asp Phe Ile Leu Met Ser Thr Ser Glu Glu Asn Lys Val 1745 1750 1755 1760Ser Gln Val Lys Ile Arg Phe Val Asn Val Phe Lys Asp Lys Thr Leu 1765 1770 1775 Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys Gln Asp Val Pro Val 1780 1785 1790 Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser Tyr Tyr Glu Asp Gly Leu 1795 1800 1805 Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu Tyr Asn Glu Lys Phe Tyr 1810 1815 1820 Ile Asn Asn Phe Gly Met Met Val Ser Gly Leu Ile Tyr Ile Asn Asp 1825 1830 1835 1840Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn Asn Leu Ile Thr Gly Phe 1845 1850 1855 Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro Ile Asn Gly Gly 1860 1865 1870 Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp Asp Lys Asn Tyr Tyr Phe 1875 1880 1885 Asn Gln Ser Gly Val Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly 1890 1895 1900 Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly 1905 1910 1915 1920Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr 1925 1930 1935 Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp 1940 1945 1950 Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly 1955 1960 1965 Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val 1970 1975 1980 Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr Phe Asp 1985 1990 1995 2000Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp Gly Lys His 2005 2010 2015 Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn Thr 2020 2025 2030 Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn 2035 2040 2045 Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn 2050 2055 2060 Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys 2065 2070 2075 2080Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu 2085 2090 2095 Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn 2100 2105 2110 Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn Asp Lys Val 2115 2120 2125 Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile 2130 2135 2140 Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly 2145 2150 2155 2160Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr 2165 2170 2175 Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val 2180 2185 2190 Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu 2195 2200 2205 Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe 2210 2215 2220 Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile Asp Asp 2225 2230 2235 2240Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr Gly Leu Ile 2245 2250 2255 Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gln 2260 2265 2270 Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp 2275 2280 2285 Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr 2290 2295 2300 Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile 2305 2310 2315 2320Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr 2325 2330 2335 Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu 2340 2345 2350 Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu 2355 2360 2365 532366PRTArtificial Sequence078 TcdB 53Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg 1 5 10 15 Phe Arg Thr Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu 20 25 30 Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys 35 40 45 Leu Lys Asp Ile Asn Ser Leu Thr Asp Ile Tyr Ile Asp Thr Tyr Lys 50 55 60 Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val 65 70 75 80 Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys 85 90 95 Asn Leu His Phe Val Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile

100 105 110 Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn 115 120 125 Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr 130 135 140 Ile Val Glu Ser Ala Ile Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn 145 150 155 160 Leu Asn Asn Pro Arg Phe Asp Tyr Asn Lys Phe Phe Arg Lys Arg Met 165 170 175 Glu Ile Ile Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Ala 180 185 190 Gln Arg Glu Glu Asn Pro Glu Leu Ile Ile Asp Asp Ile Val Lys Ile 195 200 205 Tyr Leu Ser Asn Glu Tyr Ser Lys Glu Ile Asp Glu Leu Asn Thr Tyr 210 215 220 Ile Glu Glu Ser Leu Asn Lys Ile Lys Gln Asn Ser Gly Asn Asp Val 225 230 235 240 Arg Asn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Lys Leu Tyr Glu 245 250 255 Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu 260 265 270 Arg Ile Ser Ala Leu Lys Glu Ile Gly Gly Met Tyr Leu Asp Val Asp 275 280 285 Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro 290 295 300 Ser Ser Val Thr Val Asp Phe Trp Glu Met Thr Lys Leu Glu Ala Ile 305 310 315 320 Met Lys Tyr Lys Glu Tyr Ile Pro Gly Tyr Thr Ser Glu His Phe Asp 325 330 335 Met Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Ala Leu Ala Ser 340 345 350 Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Met Glu Ala 355 360 365 Ser Pro Leu Glu Val Lys Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn 370 375 380 Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val 385 390 395 400 Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro 405 410 415 Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe Ile 420 425 430 Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met 435 440 445 Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr 450 455 460 Thr Val Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp 465 470 475 480 Leu Leu Met Phe Lys Glu Gly Ser Met Asn Ile His Leu Ile Glu Ala 485 490 495 Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr 500 505 510 Glu Gln Glu Met Ala Ser Leu Trp Thr Phe Asp Asp Ala Arg Ala Lys 515 520 525 Val Gln Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ser Leu Gly 530 535 540 Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Ile Val Val Asp Lys Glu 545 550 555 560 Tyr Leu Leu Glu Lys Ile Ser Ser Leu Ala Arg Ser Ser Glu Arg Gly 565 570 575 Tyr Ile His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr Glu 580 585 590 Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Ile Leu Phe 595 600 605 Gln Lys Asn Ile Glu Asn Ser Glu Val Ala Tyr Tyr Tyr Asn Pro Gly 610 615 620 Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Arg Ile Pro Ser Ile Ile 625 630 635 640 Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp 645 650 655 Glu Phe Asn Thr Asp Ile Phe Ala Gly Leu Asp Val Asp Ser Leu Ser 660 665 670 Thr Glu Ile Glu Thr Ala Ile Asp Leu Ala Lys Glu Asp Ile Ser Ser 675 680 685 Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser 690 695 700 Ile Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys 705 710 715 720 Asp Lys Ile Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile Ile 725 730 735 Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Asn Glu Gly Arg Arg 740 745 750 Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile 755 760 765 Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys Glu 770 775 780 Asn Lys Ile Thr Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu 785 790 795 800 Leu Gln Glu Ile Arg Asn Asn Ser Asn Leu Ser Asp Ile Glu Leu Glu 805 810 815 Glu Lys Val Met Leu Ala Glu Cys Glu Ile Asn Val Ile Ser Asn Ile 820 825 830 Asp Thr Gln Ile Val Glu Glu Arg Ile Glu Glu Ala Lys Asn Leu Thr 835 840 845 Ser Asp Ser Ile Asn Tyr Ile Lys Asn Glu Phe Lys Leu Ile Glu Ser 850 855 860 Ile Ser Asp Ser Leu Tyr Asp Leu Lys Gln Gln Asn Glu Leu Asp Asp 865 870 875 880 Ser His Phe Ile Ser Phe Glu Asp Ile Ser Lys Thr Glu Asp Gly Phe 885 890 895 Ser Ile Arg Phe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val Glu 900 905 910 Thr Glu Lys Glu Ile Phe Ser Glu Tyr Ala Asn His Ile Glu Arg Glu 915 920 925 Ile Ser Asn Ile Lys Asp Thr Ile Phe Asp Thr Val Asn Gly Lys Leu 930 935 940 Val Lys Lys Val Asn Leu Asp Ala Ile His Glu Val Asn Thr Leu Asn 945 950 955 960 Ala Ala Phe Phe Ile Gln Ser Leu Ile Gly Tyr Ser Ser Ser Lys Glu 965 970 975 Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln 980 985 990 Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val Val 995 1000 1005 Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro Thr 1010 1015 1020 Leu Ser Glu Gly Leu Pro Val Ile Ala Thr Ile Ile Asp Gly Val Ser 1025 1030 1035 1040Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser Asp Pro Leu Leu 1045 1050 1055 Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala Val Asn Leu Thr 1060 1065 1070 Ala Ala Thr Thr Ala Ile Ile Thr Ser Ser Leu Gly Ile Ala Ser Gly 1075 1080 1085 Phe Ser Ile Leu Leu Val Pro Leu Ala Gly Ile Ser Ala Gly Ile Pro 1090 1095 1100 Ser Leu Val Asn Asn Glu Leu Val Leu Arg Asp Lys Ala Thr Lys Val 1105 1110 1115 1120Val Asp Tyr Phe Lys His Ile Ser Leu Val Glu Thr Glu Gly Ala Phe 1125 1130 1135 Thr Leu Leu Asp Asp Lys Ile Met Ile Pro Gln Asp Asp Leu Val Ile 1140 1145 1150 Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Val Leu Gly Lys Cys Glu 1155 1160 1165 Ile Trp Arg Met Glu Gly Gly Ser Gly His Thr Val Thr Asn Asp Ile 1170 1175 1180 Asp His Phe Phe Ser Ser Pro Thr Ile Thr Tyr Ile Lys Pro His Leu 1185 1190 1195 1200Ser Ile Tyr Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser 1205 1210 1215 Lys Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp 1220 1225 1230 Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Glu Gly Thr 1235 1240 1245 Lys Leu Leu Asp Arg Ile Arg Asp His Tyr Lys Gly Glu Phe Tyr Trp 1250 1255 1260 Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr Leu Lys Pro 1265 1270 1275 1280Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp Ser Asn Asn Arg 1285 1290 1295 Ser Phe Ile Val Pro Ile Ile Thr Thr Glu His Ile Arg Glu Lys Leu 1300 1305 1310 Ser Tyr Ser Phe His Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu Ser 1315 1320 1325 Gln Tyr Asn Met Gly Ile Asn Ile Glu Leu Ser Glu Ser Asp Val Trp 1330 1335 1340 Ile Ile Asp Val Asp Asn Val Val Arg Asp Val Thr Ile Asp Ser Asp 1345 1350 1355 1360Lys Ile Lys Lys Gly Asp Leu Ile Glu Gly Ile Leu Ser Thr Leu Ser 1365 1370 1375 Ile Glu Asp Asn Lys Ile Ile Leu Asn His His Glu Ile Asn Phe Ser 1380 1385 1390 Gly Asp Val Asn Gly Ser Asn Gly Phe Ile Ser Leu Thr Phe Ser Ile 1395 1400 1405 Leu Glu Gly Ile Asn Ala Ile Ile Glu Val Asp Leu Leu Ser Lys Ser 1410 1415 1420 Tyr Lys Leu Leu Ile Ser Gly Glu Leu Lys Ile Leu Met Leu Asn Ser 1425 1430 1435 1440Asn His Ile Gln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu 1445 1450 1455 Gln Lys Asn Ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn 1460 1465 1470 Gly Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu 1475 1480 1485 Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser Lys Pro 1490 1495 1500 Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys Val Ile Thr 1505 1510 1515 1520Lys Asp Asn Val Asn Ile Leu Thr Gly Tyr Tyr Leu Lys Asp Asp Ile 1525 1530 1535 Lys Ile Ser Leu Ser Phe Thr Leu Gln Asp Glu Lys Thr Ile Lys Leu 1540 1545 1550 Asn Gly Val His Leu Asp Glu Ser Gly Val Ala Glu Ile Leu Lys Phe 1555 1560 1565 Met Asn Lys Lys Gly Ser Thr Asn Thr Ser Asp Ser Leu Met Ser Phe 1570 1575 1580 Leu Glu Ser Val Asn Ile Lys Ser Ile Phe Val Asn Phe Leu Gln Ser 1585 1590 1595 1600Lys Ile Asn Phe Ile Leu Asp Ala Asn Phe Ile Ile Ser Gly Thr Thr 1605 1610 1615 Ser Ile Gly Gln Phe Glu Phe Ile Cys Asp Glu Asn Asp Asn Ile Gln 1620 1625 1630 Pro Tyr Phe Ile Lys Phe Asn Thr Leu Glu Thr Thr Tyr Thr Leu Tyr 1635 1640 1645 Val Gly Asn Arg Gln Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp 1650 1655 1660 Asp Ser Gly Asp Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr 1665 1670 1675 1680Leu Tyr Gly Ile Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro Asn 1685 1690 1695 Ile Tyr Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Thr Asn Asn 1700 1705 1710 Asn Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu Lys 1715 1720 1725 Ile Asn Val Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser Asn 1730 1735 1740 Asp Gly Asn Asp Phe Ile Leu Met Ser Thr Ser Glu Glu Asn Lys Val 1745 1750 1755 1760Ser Gln Val Lys Ile Arg Phe Val Asn Val Phe Lys Asp Lys Thr Leu 1765 1770 1775 Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys Gln Asp Val Pro Val 1780 1785 1790 Ser Glu Ile Ile Ser Ala Phe Thr Pro Ser Tyr Tyr Glu Asp Gly Leu 1795 1800 1805 Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu Tyr Asn Glu Lys Phe Tyr 1810 1815 1820 Ile Asn Asn Phe Gly Met Met Val Ser Gly Leu Ile Tyr Ile Asn Asp 1825 1830 1835 1840Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn Asn Leu Ile Thr Gly Phe 1845 1850 1855 Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro Thr Asn Gly Gly 1860 1865 1870 Ala Ala Ser Ile Gly Glu Thr Ile Ile Asn Asp Lys Asn Tyr Tyr Phe 1875 1880 1885 Asn Gln Ser Gly Ile Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly 1890 1895 1900 Leu Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly 1905 1910 1915 1920Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr 1925 1930 1935 Tyr Phe Glu Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp 1940 1945 1950 Gly Glu Met Tyr Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly 1955 1960 1965 Leu Asn Gln Ile Gly Asp Asp Lys Tyr Tyr Phe Asn Ser Asp Gly Ile 1970 1975 1980 Met Gln Lys Gly Phe Val Ser Ile Asn Asp Lys Lys Tyr Tyr Phe Asp 1985 1990 1995 2000Asp Ser Gly Val Met Lys Val Gly Tyr Ile Glu Ile Asp Gly Lys Tyr 2005 2010 2015 Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn Thr 2020 2025 2030 Ser Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn 2035 2040 2045 Glu Glu Gly Glu Ala Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn 2050 2055 2060 Lys Ile Tyr Tyr Phe Asp Tyr Ser Phe Thr Ala Val Val Gly Trp Lys 2065 2070 2075 2080Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu 2085 2090 2095 Ala Tyr Val Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn 2100 2105 2110 Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn Asn Lys Val 2115 2120 2125 Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile 2130 2135 2140 Asp Asp Asn Tyr Phe Tyr Ile Asp Glu Lys Gly Ile Val Gln Ile Gly 2145 2150 2155 2160Val Phe Asp Thr Ser Asp Glu Tyr Lys Tyr Phe Ala Pro Ala Asn Thr 2165 2170 2175 Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Asp Tyr Ser Gly Leu Val 2180 2185 2190 Arg Val Gly Glu Asp Ile Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu 2195 2200 2205 Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe 2210 2215 2220 Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile Asp Asp 2225 2230 2235 2240Ile Lys Tyr Tyr Phe Asp Glu Asn Gly Ile Met Arg Thr Gly Leu Ile 2245 2250 2255 Ser Phe Glu Asn Asn Asp Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gln 2260 2265 2270 Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp 2275 2280 2285 Gly Val Met Gln Ile Gly Val Phe Asn Thr Gln Asp Gly Phe Lys Tyr 2290 2295 2300 Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile 2305 2310 2315 2320Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr 2325 2330 2335 Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu 2340 2345 2350 Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu 2355 2360 2365 542367PRTArtificial Sequence017 TcdB 54Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg 1 5 10 15 Phe Arg Val Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu 20 25 30 Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu

Lys 35 40 45 Leu Lys Asp Ile Asn Ser Leu Thr Asp Thr Tyr Ile Asp Thr Tyr Lys 50 55 60 Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val 65 70 75 80 Ile Glu Ile Leu Glu Leu Lys Asn Ser Asn Leu Thr Pro Val Glu Lys 85 90 95 Asn Leu His Phe Ile Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile 100 105 110 Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn 115 120 125 Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr 130 135 140 Ile Ile Glu Ser Ala Ser Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn 145 150 155 160 Leu Asn Asp Pro Glu Phe Asn His Thr Ala Phe Phe Arg Lys Arg Met 165 170 175 Gln Ile Ile Tyr Asp Lys Gln Gln Asn Phe Ile Asn Tyr Tyr Lys Ala 180 185 190 Gln Lys Glu Glu Asn Pro Asp Leu Ile Ile Asp Asp Ile Val Lys Thr 195 200 205 Tyr Leu Ser Asn Glu Tyr Ser Lys Asp Ile Asp Glu Leu Asn Ala Tyr 210 215 220 Ile Glu Glu Ser Leu Asn Lys Val Thr Glu Asn Ser Gly Asn Asp Val 225 230 235 240 Arg Asn Phe Glu Glu Phe Lys Thr Gly Glu Val Phe Asn Leu Tyr Glu 245 250 255 Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Gly Ala Ser Asp Ile Leu 260 265 270 Arg Val Ala Ile Leu Lys Asn Ile Gly Gly Val Tyr Leu Asp Val Asp 275 280 285 Met Leu Pro Gly Ile His Pro Asp Leu Phe Lys Asp Ile Asn Lys Pro 290 295 300 Asp Ser Val Lys Thr Ala Val Asp Trp Glu Glu Met Gln Leu Glu Ala 305 310 315 320 Ile Met Lys His Lys Glu Tyr Ile Pro Glu Tyr Thr Ser Lys His Phe 325 330 335 Asp Thr Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala 340 345 350 Ser Lys Ser Asp Lys Ser Glu Ile Phe Leu Pro Leu Gly Asp Ile Glu 355 360 365 Val Ser Pro Leu Glu Val Lys Ile Ala Phe Ala Lys Gly Ser Ile Ile 370 375 380 Asn Gln Ala Leu Ile Ser Ala Lys Asp Ser Tyr Cys Ser Asp Leu Leu 385 390 395 400 Ile Lys Gln Ile Gln Asn Arg Tyr Lys Ile Leu Asn Asp Thr Leu Gly 405 410 415 Pro Ile Ile Ser Gln Gly Asn Asp Phe Asn Thr Thr Met Asn Asn Phe 420 425 430 Gly Glu Ser Leu Gly Ala Ile Ala Asn Glu Glu Asn Ile Ser Phe Ile 435 440 445 Ala Lys Ile Gly Ser Tyr Leu Arg Val Gly Phe Tyr Pro Glu Ala Asn 450 455 460 Thr Thr Ile Thr Leu Ser Gly Pro Thr Ile Tyr Ala Gly Ala Tyr Lys 465 470 475 480 Asp Leu Leu Thr Phe Lys Glu Met Ser Ile Asp Thr Ser Ile Leu Ser 485 490 495 Ser Glu Leu Arg Asn Phe Glu Phe Pro Lys Val Asn Ile Ser Gln Ala 500 505 510 Thr Glu Gln Glu Lys Asn Ser Leu Trp Gln Phe Asn Glu Glu Arg Ala 515 520 525 Lys Ile Gln Phe Glu Glu Tyr Lys Lys Asn Tyr Phe Glu Gly Ala Leu 530 535 540 Gly Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Thr Val Thr Asp Lys 545 550 555 560 Glu Tyr Leu Leu Glu Lys Ile Ser Ser Ser Thr Lys Ser Ser Glu Arg 565 570 575 Gly Tyr Val His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr 580 585 590 Glu Ala Ala Cys Asn Leu Phe Ala Lys Asn Pro Tyr Asp Ser Ile Leu 595 600 605 Phe Gln Lys Asn Ile Glu Asp Ser Glu Val Ala Tyr Tyr Tyr Asn Pro 610 615 620 Thr Asp Ser Glu Ile Gln Glu Ile Asp Lys Tyr Arg Ile Pro Asp Arg 625 630 635 640 Ile Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys 645 650 655 Ala Glu Phe Asn Thr Asp Ile Phe Ala Gly Leu Asp Val Asp Ser Leu 660 665 670 Ser Ser Glu Ile Glu Thr Ala Ile Gly Leu Ala Lys Glu Asp Ile Ser 675 680 685 Pro Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr 690 695 700 Ser Val Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Arg Val 705 710 715 720 Lys Asp Lys Val Ser Glu Leu Met Pro Ser Met Ser Gln Asp Ser Ile 725 730 735 Ile Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg 740 745 750 Arg Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser 755 760 765 Ile Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys 770 775 780 Glu Asn Lys Ile Ile Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr 785 790 795 800 Leu Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp Ile Glu Leu 805 810 815 Glu Glu Lys Val Met Leu Ala Glu Cys Glu Ile Asn Val Ile Ser Asn 820 825 830 Ile Glu Thr Gln Val Val Glu Glu Arg Ile Glu Glu Ala Lys Ser Leu 835 840 845 Thr Ser Asp Ser Ile Asn Tyr Ile Lys Asn Glu Phe Lys Leu Ile Glu 850 855 860 Ser Ile Ser Asp Ala Leu Cys Asp Leu Lys Gln Gln Asn Glu Leu Glu 865 870 875 880 Asp Ser His Phe Ile Ser Phe Glu Asp Ile Ser Glu Thr Asp Glu Gly 885 890 895 Phe Ser Ile Arg Phe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val 900 905 910 Glu Thr Glu Lys Thr Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu 915 920 925 Glu Ile Ser Lys Ile Lys Gly Thr Ile Phe Asp Thr Val Asn Gly Lys 930 935 940 Leu Val Lys Lys Val Asn Leu Asp Thr Thr His Glu Val Asn Thr Leu 945 950 955 960 Asn Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr Asn Ser Ser Lys 965 970 975 Glu Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala 980 985 990 Gln Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val 995 1000 1005 Val Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro 1010 1015 1020 Thr Leu Ser Glu Gly Leu Pro Ile Ile Ala Thr Ile Ile Asp Gly Val 1025 1030 1035 1040Ser Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser Asp Pro Leu 1045 1050 1055 Leu Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala Val Asn Leu 1060 1065 1070 Thr Thr Ala Thr Thr Ala Ile Ile Thr Ser Ser Leu Gly Ile Ala Ser 1075 1080 1085 Gly Phe Ser Ile Leu Leu Val Pro Leu Ala Gly Ile Ser Ala Gly Ile 1090 1095 1100 Pro Ser Leu Val Asn Asn Glu Leu Val Leu Arg Asp Lys Ala Thr Lys 1105 1110 1115 1120Val Val Asp Tyr Phe Lys His Val Ser Leu Val Glu Thr Glu Gly Val 1125 1130 1135 Phe Thr Leu Leu Asp Asp Lys Val Met Met Pro Gln Asp Asp Leu Val 1140 1145 1150 Ile Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Val Leu Gly Lys Cys 1155 1160 1165 Glu Ile Trp Arg Met Glu Gly Gly Ser Gly His Thr Val Thr Asp Asp 1170 1175 1180 Ile Asp His Phe Phe Ser Ala Pro Ser Ile Thr Tyr Arg Glu Pro His 1185 1190 1195 1200Leu Ser Ile Tyr Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu 1205 1210 1215 Ser Lys Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala 1220 1225 1230 Trp Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly 1235 1240 1245 Thr Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu Gly Glu Phe Tyr 1250 1255 1260 Trp Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr Leu Lys 1265 1270 1275 1280Pro Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp Ser Asn Thr 1285 1290 1295 Arg Ser Phe Ile Val Pro Ile Ile Thr Thr Glu Tyr Ile Arg Glu Lys 1300 1305 1310 Leu Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu 1315 1320 1325 Ser Gln Tyr Asn Met Gly Ile Asn Ile Glu Leu Ser Glu Ser Asp Val 1330 1335 1340 Trp Ile Ile Asp Val Asp Asn Val Val Arg Asp Val Thr Ile Glu Ser 1345 1350 1355 1360Asp Lys Ile Lys Lys Gly Asp Leu Ile Glu Gly Ile Leu Ser Thr Leu 1365 1370 1375 Ser Ile Glu Glu Asn Lys Ile Ile Leu Asn Ser His Glu Ile Asn Phe 1380 1385 1390 Ser Gly Glu Val Asn Gly Ser Asn Gly Phe Val Ser Leu Thr Phe Ser 1395 1400 1405 Ile Leu Glu Gly Ile Asn Ala Ile Ile Glu Val Asp Leu Leu Ser Lys 1410 1415 1420 Ser Tyr Lys Leu Leu Ile Ser Gly Glu Leu Lys Ile Leu Met Leu Asn 1425 1430 1435 1440Ser Asn His Ile Gln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu 1445 1450 1455 Leu Gln Lys Asn Ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu 1460 1465 1470 Asn Gly Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu 1475 1480 1485 Leu Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser Lys 1490 1495 1500 Pro Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys Val Ile 1505 1510 1515 1520Thr Lys Asp Asn Val Asn Ile Leu Thr Gly Tyr Tyr Leu Lys Asp Asp 1525 1530 1535 Ile Lys Ile Ser Leu Ser Leu Thr Leu Gln Asp Glu Lys Thr Ile Lys 1540 1545 1550 Leu Asn Ser Val His Leu Asp Glu Ser Gly Val Ala Glu Ile Leu Lys 1555 1560 1565 Phe Met Asn Arg Lys Gly Ser Thr Asn Thr Ser Asp Ser Leu Met Ser 1570 1575 1580 Phe Leu Glu Ser Met Asn Ile Lys Ser Ile Phe Val Asn Phe Leu Gln 1585 1590 1595 1600Ser Asn Ile Lys Phe Ile Leu Asp Ala Asn Phe Ile Ile Ser Gly Thr 1605 1610 1615 Thr Ser Ile Gly Gln Phe Glu Phe Ile Cys Asp Glu Asn Asn Asn Ile 1620 1625 1630 Gln Pro Tyr Phe Ile Lys Phe Asn Thr Leu Glu Thr Asn Tyr Thr Leu 1635 1640 1645 Tyr Val Gly Asn Arg Gln Asn Met Ile Val Glu Pro Asn Tyr Asp Leu 1650 1655 1660 Asp Asp Ser Gly Asp Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys 1665 1670 1675 1680Tyr Leu Tyr Gly Ile Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro 1685 1690 1695 Asn Ile Tyr Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Thr Asn 1700 1705 1710 Asn Thr Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu 1715 1720 1725 Lys Ile Asn Val Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser 1730 1735 1740 Asn Asp Gly Asn Asp Phe Ile Leu Met Ser Thr Ser Glu Glu Asn Lys 1745 1750 1755 1760Val Ser Gln Val Lys Ile Arg Phe Val Asn Val Phe Lys Asp Lys Thr 1765 1770 1775 Leu Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys Gln Asp Val Pro 1780 1785 1790 Val Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser Tyr Tyr Glu Asp Gly 1795 1800 1805 Leu Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu Tyr Asn Glu Lys Phe 1810 1815 1820 Tyr Ile Asn Asn Phe Gly Met Met Val Ser Gly Leu Ile Tyr Ile Asn 1825 1830 1835 1840Asp Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn Asn Leu Ile Thr Gly 1845 1850 1855 Phe Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro Ile Asn Gly 1860 1865 1870 Gly Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp Asp Lys Asn Tyr Tyr 1875 1880 1885 Phe Asn Gln Ser Gly Val Leu Gln Thr Gly Val Phe Ser Thr Glu Asp 1890 1895 1900 Gly Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu 1905 1910 1915 1920Gly Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile 1925 1930 1935 Tyr Tyr Phe Glu Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu 1940 1945 1950 Asp Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys 1955 1960 1965 Gly Leu Asn Gln Ile Gly Asp Asp Lys Tyr Tyr Phe Asn Ser Asp Gly 1970 1975 1980 Val Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr Phe 1985 1990 1995 2000Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp Gly Lys 2005 2010 2015 His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn 2020 2025 2030 Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly 2035 2040 2045 Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn 2050 2055 2060 Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp 2065 2070 2075 2080Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala 2085 2090 2095 Glu Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe 2100 2105 2110 Asn Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn Asp Lys 2115 2120 2125 Val Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn 2130 2135 2140 Ile Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile 2145 2150 2155 2160Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn 2165 2170 2175 Thr Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu 2180 2185 2190 Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile 2195 2200 2205 Glu Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr 2210 2215 2220 Phe Asp Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile Asp 2225 2230 2235 2240Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr Gly Leu 2245 2250 2255 Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met 2260 2265 2270 Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly Glu 2275 2280 2285 Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro Asp Gly Phe Lys 2290 2295 2300 Tyr Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser 2305 2310 2315 2320Ile Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe 2325 2330 2335 Thr Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu 2340

2345 2350 Glu Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu 2355 2360 2365 552366PRTArtificial Sequence027 TcdB 55Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg 1 5 10 15 Phe Arg Val Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu 20 25 30 Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys 35 40 45 Leu Lys Asp Ile Asn Ser Leu Thr Asp Ile Tyr Ile Asp Thr Tyr Lys 50 55 60 Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val 65 70 75 80 Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys 85 90 95 Asn Leu His Phe Val Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile 100 105 110 Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn 115 120 125 Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr 130 135 140 Ile Val Glu Ser Ala Thr Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn 145 150 155 160 Leu Asn Asp Pro Arg Phe Asp Tyr Asn Lys Phe Tyr Arg Lys Arg Met 165 170 175 Glu Ile Ile Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Thr 180 185 190 Gln Arg Glu Glu Asn Pro Asp Leu Ile Ile Asp Asp Ile Val Lys Ile 195 200 205 Tyr Leu Ser Asn Glu Tyr Ser Lys Asp Ile Asp Glu Leu Asn Ser Tyr 210 215 220 Ile Glu Glu Ser Leu Asn Lys Val Thr Glu Asn Ser Gly Asn Asp Val 225 230 235 240 Arg Asn Phe Glu Glu Phe Lys Gly Gly Glu Ser Phe Lys Leu Tyr Glu 245 250 255 Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu 260 265 270 Arg Ile Ser Ala Leu Lys Glu Val Gly Gly Val Tyr Leu Asp Val Asp 275 280 285 Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro 290 295 300 Ser Ser Val Thr Val Asp Phe Trp Glu Met Val Lys Leu Glu Ala Ile 305 310 315 320 Met Lys Tyr Lys Glu Tyr Ile Pro Gly Tyr Thr Ser Glu His Phe Asp 325 330 335 Met Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala Ser 340 345 350 Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Met Glu Ala 355 360 365 Ser Pro Leu Glu Val Lys Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn 370 375 380 Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val 385 390 395 400 Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro 405 410 415 Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Ala Phe Ile 420 425 430 Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met 435 440 445 Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr 450 455 460 Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp 465 470 475 480 Leu Leu Met Phe Lys Glu Gly Ser Met Asn Ile His Leu Ile Glu Ala 485 490 495 Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr 500 505 510 Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys 515 520 525 Ala Gln Phe Glu Glu Tyr Lys Lys Asn Tyr Phe Glu Gly Ser Leu Gly 530 535 540 Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Thr Val Val Asp Lys Glu 545 550 555 560 Tyr Leu Leu Glu Lys Ile Ser Ser Leu Ala Arg Ser Ser Glu Arg Gly 565 570 575 Tyr Ile His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr Glu 580 585 590 Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Val Leu Phe 595 600 605 Gln Lys Asn Ile Glu Asp Ser Glu Ile Ala Tyr Tyr Tyr Asn Pro Gly 610 615 620 Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Lys Ile Pro Ser Ile Ile 625 630 635 640 Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp 645 650 655 Glu Phe Asn Thr Asp Ile Phe Ala Gly Leu Asp Val Asp Ser Leu Ser 660 665 670 Thr Glu Ile Glu Thr Ala Ile Asp Leu Ala Lys Glu Asp Ile Ser Pro 675 680 685 Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser 690 695 700 Val Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Arg Val Lys 705 710 715 720 Asp Lys Val Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile Ile 725 730 735 Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg 740 745 750 Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile 755 760 765 Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys Glu 770 775 780 Asn Lys Ile Ile Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu 785 790 795 800 Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp Ile Glu Leu Glu 805 810 815 Glu Lys Val Met Leu Ala Glu Cys Glu Ile Asn Val Ile Ser Asn Ile 820 825 830 Asp Thr Gln Val Val Glu Gly Arg Ile Glu Glu Ala Lys Ser Leu Thr 835 840 845 Ser Asp Ser Ile Asn Tyr Ile Lys Asn Glu Phe Lys Leu Ile Glu Ser 850 855 860 Ile Ser Asp Ala Leu Tyr Asp Leu Lys Gln Gln Asn Glu Leu Glu Glu 865 870 875 880 Ser His Phe Ile Ser Phe Glu Asp Ile Leu Glu Thr Asp Glu Gly Phe 885 890 895 Ser Ile Arg Phe Ile Asp Lys Glu Thr Gly Glu Ser Ile Phe Val Glu 900 905 910 Thr Glu Lys Ala Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu Glu 915 920 925 Ile Ser Lys Ile Lys Gly Thr Ile Phe Asp Thr Val Asn Gly Lys Leu 930 935 940 Val Lys Lys Val Asn Leu Asp Ala Thr His Glu Val Asn Thr Leu Asn 945 950 955 960 Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr Asn Ser Ser Lys Glu 965 970 975 Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln 980 985 990 Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val Val 995 1000 1005 Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro Thr 1010 1015 1020 Leu Ser Glu Gly Leu Pro Val Ile Ala Thr Ile Ile Asp Gly Val Ser 1025 1030 1035 1040Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser Asp Pro Leu Leu 1045 1050 1055 Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala Val Asn Leu Thr 1060 1065 1070 Ala Ala Thr Thr Ala Ile Ile Thr Ser Ser Leu Gly Ile Ala Ser Gly 1075 1080 1085 Phe Ser Ile Leu Leu Val Pro Leu Ala Gly Ile Ser Ala Gly Ile Pro 1090 1095 1100 Ser Leu Val Asn Asn Glu Leu Ile Leu Arg Asp Lys Ala Thr Lys Val 1105 1110 1115 1120Val Asp Tyr Phe Ser His Ile Ser Leu Ala Glu Ser Glu Gly Ala Phe 1125 1130 1135 Thr Ser Leu Asp Asp Lys Ile Met Met Pro Gln Asp Asp Leu Val Ile 1140 1145 1150 Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Thr Leu Gly Lys Cys Glu 1155 1160 1165 Ile Trp Arg Met Glu Gly Gly Ser Gly His Thr Val Thr Asp Asp Ile 1170 1175 1180 Asp His Phe Phe Ser Ala Pro Ser Ile Thr Tyr Arg Glu Pro His Leu 1185 1190 1195 1200Ser Ile Tyr Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser 1205 1210 1215 Lys Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp 1220 1225 1230 Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr 1235 1240 1245 Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu Gly Glu Phe Tyr Trp 1250 1255 1260 Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr Leu Lys Pro 1265 1270 1275 1280Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp Ser Asn Thr Arg 1285 1290 1295 Ser Phe Ile Val Pro Val Ile Thr Thr Glu Tyr Ile Arg Glu Lys Leu 1300 1305 1310 Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu Ser 1315 1320 1325 Gln Tyr Asn Met Asn Ile Asn Ile Glu Leu Asn Glu Asn Asp Thr Trp 1330 1335 1340 Val Ile Asp Val Asp Asn Val Val Arg Asp Val Thr Ile Glu Ser Asp 1345 1350 1355 1360Lys Ile Lys Lys Gly Asp Leu Ile Glu Asn Ile Leu Ser Lys Leu Ser 1365 1370 1375 Ile Glu Asp Asn Lys Ile Ile Leu Asp Asn His Glu Ile Asn Phe Ser 1380 1385 1390 Gly Thr Leu Asn Gly Gly Asn Gly Phe Val Ser Leu Thr Phe Ser Ile 1395 1400 1405 Leu Glu Gly Ile Asn Ala Val Ile Glu Val Asp Leu Leu Ser Lys Ser 1410 1415 1420 Tyr Lys Val Leu Ile Ser Gly Glu Leu Lys Thr Leu Met Ala Asn Ser 1425 1430 1435 1440Asn Ser Val Gln Gln Lys Ile Asp Tyr Ile Gly Leu Asn Ser Glu Leu 1445 1450 1455 Gln Lys Asn Ile Pro Tyr Ser Phe Met Asp Asp Lys Gly Lys Glu Asn 1460 1465 1470 Gly Phe Ile Asn Cys Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu 1475 1480 1485 Ser Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asn Ser Lys Pro 1490 1495 1500 Leu Phe Gly Tyr Cys Ser Asn Asp Leu Lys Asp Val Lys Val Ile Thr 1505 1510 1515 1520Lys Asp Asp Val Ile Ile Leu Thr Gly Tyr Tyr Leu Lys Asp Asp Ile 1525 1530 1535 Lys Ile Ser Leu Ser Phe Thr Ile Gln Asp Glu Asn Thr Ile Lys Leu 1540 1545 1550 Asn Gly Val Tyr Leu Asp Glu Asn Gly Val Ala Glu Ile Leu Lys Phe 1555 1560 1565 Met Asn Lys Lys Gly Ser Thr Asn Thr Ser Asp Ser Leu Met Ser Phe 1570 1575 1580 Leu Glu Ser Met Asn Ile Lys Ser Ile Phe Ile Asn Ser Leu Gln Ser 1585 1590 1595 1600Asn Thr Lys Leu Ile Leu Asp Thr Asn Phe Ile Ile Ser Gly Thr Thr 1605 1610 1615 Ser Ile Gly Gln Phe Glu Phe Ile Cys Asp Lys Asp Asn Asn Ile Gln 1620 1625 1630 Pro Tyr Phe Ile Lys Phe Asn Thr Leu Glu Thr Lys Tyr Thr Leu Tyr 1635 1640 1645 Val Gly Asn Arg Gln Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp 1650 1655 1660 Asp Ser Gly Asp Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr 1665 1670 1675 1680Leu Tyr Gly Ile Asp Ser Cys Val Asn Lys Val Ile Ile Ser Pro Asn 1685 1690 1695 Ile Tyr Thr Asp Glu Ile Asn Ile Thr Pro Ile Tyr Glu Ala Asn Asn 1700 1705 1710 Thr Tyr Pro Glu Val Ile Val Leu Asp Thr Asn Tyr Ile Ser Glu Lys 1715 1720 1725 Ile Asn Ile Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser Asn 1730 1735 1740 Asp Gly Ser Asp Phe Ile Leu Met Ser Thr Asp Glu Glu Asn Lys Val 1745 1750 1755 1760Ser Gln Val Lys Ile Arg Phe Thr Asn Val Phe Lys Gly Asn Thr Ile 1765 1770 1775 Ser Asp Lys Ile Ser Phe Asn Phe Ser Asp Lys Gln Asp Val Ser Ile 1780 1785 1790 Asn Lys Val Ile Ser Thr Phe Thr Pro Ser Tyr Tyr Val Glu Gly Leu 1795 1800 1805 Leu Asn Tyr Asp Leu Gly Leu Ile Ser Leu Tyr Asn Glu Lys Phe Tyr 1810 1815 1820 Ile Asn Asn Phe Gly Met Met Val Ser Gly Leu Val Tyr Ile Asn Asp 1825 1830 1835 1840Ser Leu Tyr Tyr Phe Lys Pro Pro Ile Lys Asn Leu Ile Thr Gly Phe 1845 1850 1855 Thr Thr Ile Gly Asp Asp Lys Tyr Tyr Phe Asn Pro Asp Asn Gly Gly 1860 1865 1870 Ala Ala Ser Val Gly Glu Thr Ile Ile Asp Gly Lys Asn Tyr Tyr Phe 1875 1880 1885 Ser Gln Asn Gly Val Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly 1890 1895 1900 Phe Lys Tyr Phe Ala Pro Ala Asp Thr Leu Asp Glu Asn Leu Glu Gly 1905 1910 1915 1920Glu Ala Ile Asp Phe Thr Gly Lys Leu Thr Ile Asp Glu Asn Val Tyr 1925 1930 1935 Tyr Phe Gly Asp Asn Tyr Arg Ala Ala Ile Glu Trp Gln Thr Leu Asp 1940 1945 1950 Asp Glu Val Tyr Tyr Phe Ser Thr Asp Thr Gly Arg Ala Phe Lys Gly 1955 1960 1965 Leu Asn Gln Ile Gly Asp Asp Lys Phe Tyr Phe Asn Ser Asp Gly Ile 1970 1975 1980 Met Gln Lys Gly Phe Val Asn Ile Asn Asp Lys Thr Phe Tyr Phe Asp 1985 1990 1995 2000Asp Ser Gly Val Met Lys Ser Gly Tyr Thr Glu Ile Asp Gly Lys Tyr 2005 2010 2015 Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn Thr 2020 2025 2030 Ala Asp Gly Phe Lys Tyr Phe Ala His His Asp Glu Asp Leu Gly Asn 2035 2040 2045 Glu Glu Gly Glu Ala Leu Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn 2050 2055 2060 Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys 2065 2070 2075 2080Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu 2085 2090 2095 Ala Tyr Ile Gly Ile Ser Ile Ile Asn Asp Gly Lys Tyr Tyr Phe Asn 2100 2105 2110 Asp Ser Gly Ile Met Gln Ile Gly Phe Val Thr Ile Asn Asn Glu Val 2115 2120 2125 Phe Tyr Phe Ser Asp Ser Gly Ile Val Glu Ser Gly Met Gln Asn Ile 2130 2135 2140 Asp Asp Asn Tyr Phe Tyr Ile Asp Glu Asn Gly Leu Val Gln Ile Gly 2145 2150 2155 2160Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr 2165 2170 2175 Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val 2180 2185 2190 Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu 2195 2200 2205 Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe 2210 2215 2220 Asp Pro Glu Thr Lys Lys Ala Tyr Lys Gly Ile Asn Val Ile Asp Asp 2225 2230 2235 2240Ile Lys Tyr Tyr Phe Asp Glu Asn Gly Ile Met Arg Thr Gly Leu Ile 2245 2250 2255 Thr Phe Glu Asp Asn His Tyr Tyr Phe Asn Glu Asp Gly Ile Met Gln 2260 2265 2270 Tyr Gly Tyr Leu Asn Ile Glu Asp Lys Thr Phe Tyr Phe Ser Glu

Asp 2275 2280 2285 Gly Ile Met Gln Ile Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr 2290 2295 2300 Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile 2305 2310 2315 2320Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr 2325 2330 2335 Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu 2340 2345 2350 Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu 2355 2360 2365

* * * * *

File A Patent Application

  • Protect your idea -- Don't let someone else file first. Learn more.

  • 3 Easy Steps -- Complete Form, application Review, and File. See our process.

  • Attorney Review -- Have your application reviewed by a Patent Attorney. See what's included.