Computational design of α(v)β(6) integrin-binding proteins

Abstract

This article discloses the α(v)β(6) integrin (avb6) binding peptide and its use in the treatment and detection of tumors, as well as its use in the treatment of pulmonary fibrosis.

Description

[0001] Cross-references

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 925868, filed October 25, 2019, the entirety of which is incorporated herein by reference.

[0003] Federal Funding Explanation

[0004] This invention was completed with government funding granted by the National Institutes of Health under grant number R01 GM092802. The government has certain rights to this invention.

[0005] Sequence List Description

[0006] The computer-readable form of the sequence list is submitted electronically with this application and is incorporated herein in its entirety by reference. The sequence list is contained in a file created on October 21, 2020, named "19-1733-PCT_Sequence-Listing_ST25.txt", and is 42kb in size. Background Technology

[0007] Integrins are a class of heterodimeric cell surface proteins involved in a wide range of cellular functions, including cell-cell adhesion, migration, proliferation, and death. avb6, one such integrin, is composed of av and b6 subunits and is responsible for activating TGF-B1 / B3. avb6 expression is strictly limited to epithelial cells. Under normal physiological conditions, avb6 expression is almost entirely confined to specific tissue morphological changes during developmental stages, resulting in low or no expression in fully differentiated epithelial cells, with some exceptions. In pathological tissue reprogramming, avb6 expression is upregulated in tumor cell migration, wound healing, and inflammation. Overall, avb6 expression levels are associated with poor overall survival. Summary of the Invention

[0008] In one aspect, the present invention discloses a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 1-3, wherein the polypeptide is bound to α(v)β(6) integrin (avb6). In one embodiment, the amino acid residue at position 8 is R, the amino acid residue at position 9 is G, and the amino acid residue at position 10 is D. In various other embodiments, the amino acid residue at position 12 is A; the amino acid residue at position 13 is E or T; the amino acid residue at position 14 is L; the amino acid residue at position 15 is M, R, or K; the amino acid residue at position 16 is L; the amino acid residue at position 37 is N, S, or K; the amino acid residue at position 38 is G; wherein the amino acid residue at position 39 is A, F, or K; the amino acid residue at position 40 is E; the amino acid residue at position 61 is R or K; the amino acid residues at positions 62-67 are FP(G / R)(V / T)XT, where X is any residue listed at position 66 in Tables 1, 2, or 3, and the residues in parentheses are alternatives for that position; the amino acid residue at position 17 is R; the amino acid residue at position 36 is N; and / or the amino acid residue at position 65 is V; and / or the amino acid residue at position 67 is T.

[0009] In another embodiment, the polypeptide comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identical to the amino acid sequence of SEQ ID NO: 4-30. In one embodiment, residues 8-10 are unchanged, and optionally, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 36, 37, 38, 39, 40, 61, 62, 63, 64, 65, or 17 of the amino acid residues at positions 12, 13, 14, 15, 16, or 17 are all unchanged relative to the reference sequence. For SEQ ID NO: 4-28, residue numbering begins with the first amino acid following the optional N-terminal methionine residue, and for SEQ ID NO: 29-30, residue numbering begins with the third amino acid (Cys residue) following the optional N-terminal methionine residue.

[0010] In another aspect, the present invention provides a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with an amino acid sequence selected from SEQ ID NOs: 4-30 and 36. In one embodiment, the polypeptide has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with an amino acid sequence selected from SEQ ID NOs: 21, 25, 29, and 30. In another embodiment, the amino acid change from the reference protein is a conserved amino acid substitution. In another embodiment, the RGD sequence remains unchanged. In one embodiment, residues 8-10 are unchanged, and optionally, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 36, 37, 38, 39, 40, 61, 62, 63, 64, 65, or 17 of the amino acid residues at positions 12, 13, 14, 15, 16, or 17 are all unchanged relative to the reference sequence. For SEQ ID NO: 4-28, residue numbering begins from the first amino acid after the optional N-terminal methionine residue, and for SEQ ID NO: 29-30, residue numbering begins from the third amino acid (Cys residue) after the optional N-terminal methionine residue.

[0011] In other respects, the present invention provides nucleic acids encoding polypeptides of any embodiment or combination of embodiments disclosed herein; expression vectors comprising said nucleic acids operatively linked to a control sequence; host cells or recombinants comprising said nucleic acids or said expression vectors of any embodiment or combination of embodiments disclosed herein; and pharmaceutical compositions comprising said polypeptides, nucleic acids, expression vectors, host cells or recombinant cells and pharmaceutically acceptable vectors of any embodiment or combination of embodiments disclosed herein.

[0012] In one aspect, the present invention provides the use of the polypeptides, nucleic acids, expression vectors, host cells, recombinant cells, or pharmaceutical compositions described in any embodiment or combination of embodiments disclosed herein for any suitable purpose, including but not limited to treating and / or detecting avb6(+) tumors in vivo, blocking avb6-mediated TGF-B signaling in vitro, and treating pulmonary fibrosis such as idiopathic pulmonary fibrosis (IPF). In another aspect, the present invention provides a method for treating avb6(+) tumors or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis (IPF)), comprising administering to a subject in need a measured amount of the polypeptides, nucleic acids, expression vectors, host cells, and / or pharmaceutical compositions described in any embodiment or combination of embodiments disclosed herein, to effectively treat the subject's tumor or IPF. In yet another aspect, the present invention provides a method for detecting avb6(+) tumors, comprising administering to a subject suspected of having avb6(+) tumors a measured amount of the polypeptides, nucleic acids, expression vectors, host cells, and / or pharmaceutical compositions described herein, which are effective in detecting the subject's tumor.

[0013] In another aspect, the present invention provides a method for designing avb6-binding peptides, including the steps described in any embodiment or combination of embodiments disclosed herein and in the appendix. Attached Figure Description

[0014] Figure 1 ai.a)α v Computational design strategy for β6-binding proteins: α v a) Structure of the complex of β6 integrin (surface representation) and TGF-β1 peptide (cartoonized representation, PDB ID 4UM9). b) Low RMSD matching of the TGF-β1 peptide from the PDB database (band representation). c) Then using Rosetta... TM Non-clashing fragments were integrated into the α / β ferroredoxin fold (cartoonish representation). d) Rosetta was performed with the RGD binding loop fixed. TM Flexible sequence design. e) Then, using Rosetta TM Structural prediction identifies the design structure as a sequence of lowest energy states. f) In addition to the RGD-bound motif, two rings (ring 1 and ring 2) mediate α... v Contact with the β6 subunit. g) The typical RGD motif in the av6_3 design interacts with the receptor at the scaffold level, with Asp coordinating with Mg(II). h) The -LXXL (SEQ ID NO: 33) motif immediately following the RGD binding loop is packaged on the hydrophobic groove of the β6 subunit. i) Additional interactions mediated by loops 1 and 2, i.e., with β6 and α, respectively. vThe subunits make polar contact.

[0015] Figure 2 Figure a, site saturation mutation analysis of the designed binding compounds; Figure be, most enriched variants are charge-complementary to the receptor (see main text for details). Figures f and g, purified BP1 and BP2 against α v BLI titration of β6. Kd < 1 nM for both mutants, and each titration was performed at least twice with similar results. Figure h shows the crystal structure of BP1_disulf superimposed on the designed model (conjugate, α). v β6). Figures i and j, superimposed with crystal structure diagram k, show the design models of disulfide bonds and RGD rings. This is in contrast to α where charge reversal exists. v Compared to β8, the A39K mutation confers α v β6 specificity (β6 is Glu963, β8 is Lys902). Figure 1, for stable transfection α v β8 K562 cells were titrated with the cell surface of BP1 and BP2. BP1 cells lacking the A39K mutation titrated with approximately 7.3 nM Kd compared to α. v β8 binds, while BP2 containing the A39K mutation binds to α with a Kd concentration >500 nM. v β8 binding.

[0016] Figure 3 TMLC analysis revealed BP1 and BP2-mediated TGF-β inhibition. Both BP1 and BP2 blocked α-β with similar IC50 values ​​(199 pM and 151 pM, respectively). v β6-mediated TGF-β activation.

[0017] Figure 4 ab, Crystal structures in the first and second design strategies: a) Crystal structure of the evolutionary variant (SEQ ID NO:36) superimposed on the design model in the first design. Although the first part of the crystal structure includes RGD rings and covers well with the design model, the last helix of the fold rotates half a turn. b) For the second-generation design, the crystal structure of the previous round is superimposed on α by comparing RGD motifs. v On β6, the lengths and conformations of the two rings were sampled, and a total of 16 designs were sampled in an ordered manner in the second round.

[0018] Figure 5 The designed protein exhibits representative metal-dependent binding: the designed protein demonstrates binding with α... v Metal-dependent binding of β6. No detectable binding was observed in the absence of any metal, compared to 1 mM Ca(II) / 1 mM Mg(II) (right side). Expression or FITC fluorescence was plotted based on binding or SAPE fluorescence (Y-axis) (X-axis).

[0019] Figure 6 Using 50pM biotinylated α v β6 bound 12 clones designed in the second round to the yeast surface. Expression or FITC fluorescence was plotted on the X-axis, and binding or SAPE fluorescence was plotted on the Y-axis.

[0020] Figure 7 In the second round of design, in vitro cell surface competition detection of the five strongest binding compounds was performed. (Compared to human α-cell binding). v Compared to β8, av6_3 is more effective against human α. v β6 exhibits high selectivity. The logarithmic concentration of the conjugate (X-axis) corresponds to the average fluorescence intensity (Y-axis).

[0021] Figure 8 The classification scheme of the SSM library in av6_3. The first round of classification is at α of 200pM. v The process is performed on β6, followed by final classification using a 100pM receptor.

[0022] Figure 9 Figure a. SDS-page gel for one-step purification of BP2_disulf from cell lysates via heat treatment. Lane 1: Ladder bands; Lane 2: Crude BP2_disulf cell lysates; Lane 3: BP2_disulf cell lysates after boiling at 85°C for 10 min. Figure b. CD spectra of BP2_disulf before and after nebulization. Detailed Implementation

[0023] All references cited in this paper are incorporated herein by reference. Unless otherwise stated, the techniques used in this application can be found in several well-known references, such as: Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991, Academic Press, San Diego, CA), “Guide to Protein Purification” in Methods in Enzymology (MP Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990, Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2 nd Ed. (RI Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. EJ Murray, The Humana Press Inc., Clifton, NJ) and Ambion 1998 Catalog (Ambion, Austin, TX).

[0024] As used in this article, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise.

[0025] As used in this article, “approximately” means + / - 5% of the stated value.

[0026] All embodiments of any aspect of this invention may be used in combination unless the context clearly specifies otherwise.

[0027] Unless the context explicitly requires otherwise, throughout the specification and claims, the terms "comprise," "comprising," etc., shall be interpreted in an inclusive sense, not in an exclusive or exhaustive sense; that is, in the sense of "including but not limited to." The use of singular or plural words shall also include both the plural and singular forms, respectively. Furthermore, the terms "herein," "above," and "below," and similar meanings, as used herein, shall refer to the entirety of this application, and not to any particular part thereof.

[0028] As used herein, the amino acid residue abbreviations are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0029] In one aspect, the present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, or 3 (as shown in Table 1, Table 2, or Table 3), wherein the table shows amino acid options at each position in the polypeptide, and wherein the polypeptide binds to α(v)β(6) integrin (avb6). As disclosed in the examples herein, the inventors have engineered the claimed polypeptide as an avb6 integrin-binding protein. The engineered protein is thermostable and binds to avb6 with high affinity. The polypeptide can be used, for example, to treat and / or detect avb6(+) tumors in vivo, to block avb6-mediated TGF-B signaling in vitro, and to treat pulmonary fibrosis, for example, idiopathic pulmonary fibrosis (IPF). Examples provide saturation studies to determine the residues that may be present at each position in the polypeptide.

[0030] Table 1 / SEQ ID NO: 1 shows the amino acid residues that may be present at any position in the polypeptide using single-letter codes for amino acids, based on the saturation mutation studies described in the examples below.

[0031]

[0032]

[0033]

[0034]

[0035] Table 2 / SEQ ID NO: 2 shows the amino acid residues that may be present at any position in the polypeptide by single-letter codes for amino acids, including the more abundant mutations seen in the saturation mutation studies described in the examples below.

[0036]

[0037]

[0038]

[0039] Table 3 / SEQ ID NO: 3 shows the amino acid residues that may be present at any position in the polypeptide by single-letter codes for amino acids, including the more abundant mutations seen in the saturation mutation studies described in the examples below.

[0040]

[0041]

[0042]

[0043]

[0044] In one embodiment, the amino acid residue at position 8 is R, the amino acid residue at position 9 is G, and the amino acid residue at position 10 is D. The interface residues for avb6 binding include residues at positions 8-10, and in this embodiment, the interface residues include the RGD motif at positions 8-10.

[0045] In various other possible combinations of implementation schemes:

[0046] The 12th amino acid residue is A;

[0047] The 13th amino acid residue is either E or T;

[0048] The 14th amino acid residue is L;

[0049] The 15th amino acid residue is M, R, or K;

[0050] The 16th amino acid residue is L;

[0051] The 17th amino acid residue is R;

[0052] The 36th amino acid residue is N;

[0053] The amino acid residue at position 37 is N, S, or K;

[0054] The 38th amino acid residue is G;

[0055] The amino acid residue at position 39 is A, F, or K;

[0056] The 40th amino acid residue is E;

[0057] The 61st amino acid residue is R or K;

[0058] The amino acid residues at positions 62-67 are FP(G / R)(V / T)XT (SEQ ID NO: 35), where X is any residue listed at position 66 in Tables 1, 2 or 3, and the residues in parentheses are alternatives for that position;

[0059] The 65th amino acid residue is V; and / or

[0060] The amino acid residue at position 67 is T.

[0061] In other possible combinations of implementation schemes:

[0062] The 12th amino acid residue is A;

[0063] The 13th amino acid residue is either E or T;

[0064] The 14th amino acid residue is L;

[0065] The 15th amino acid residue is M, R, or K;

[0066] The 17th amino acid residue is R;

[0067] The 36th amino acid residue is N;

[0068] The amino acid residue at position 37 is N, S, or K;

[0069] The 38th amino acid residue is G;

[0070] The 39th amino acid residue is A, F, or K;

[0071] The 61st amino acid residue is R or K;

[0072] The amino acid residues at positions 62-67 are FP(G / R)(V / T)XT (SEQ ID NO: 35), where X is any residue listed in position 66 of Tables 1, 2 or 3, and the residues in parentheses are alternatives for that position;

[0073] The 65th amino acid residue is V; and / or

[0074] The 67th amino acid residue is T.

[0075] As described in the following examples, the amino acid residues at positions 12-17, 36-40, 61-65, and 67 of the polypeptide can directly contact avb6.

[0076] In another embodiment, the polypeptide comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identical to the amino acid sequence of SEQ ID NO: 4-28, wherein the residues in parentheses are optional. Each of these embodiment schemes includes an optional N-terminal methionine not included in SEQ ID NO: 1-3. Therefore, the residue numbering in SEQ ID NO: 4-28 begins with the first amino acid following the optional N-terminal methionine residue.

[0077] Table 4

[0078]

[0079]

[0080]

[0081] AEVRFVFRGDLTELMLRAVKDHLKKEGPHWNITSRGNELEVRGSHESDAKRIQKEFPSV QSTTQA

[0082] In other embodiments, the polypeptide comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identical to the amino acid sequence of SEQ ID NO: 29-30, wherein the residues in parentheses are optional. Each of these embodiments includes an optional N-terminal methionine and two additional N-terminal residues that are not included in SEQ ID NO: 1-3. Thus, the residue numbering in SEQ ID NO: 29-30 begins with the third amino acid (Cys residue) following the optional N-terminal methionine residue. These embodiments introduce Cys residues that allow disulfide bond binding. The introduction of disulfide bonds gives both proteins ultrathermal stability, and according to CD spectral data under non-reducing conditions, they can maintain their secondary structure at 95°C.

[0083]

[0084] In one embodiment, the polypeptide has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an amino acid sequence selected from SEQ ID NO: 21 (E13T) and SEQ ID NO: 25 (A39KG64R).

[0085] In one embodiment, residues 8-10 (residue numbering starting from the first amino acid after the optional N-terminal methionine residue) are unchanged. As described above, the interface residues for avb6 binding include residues 8-10. In another embodiment, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, sixteen, or seventeen of the amino acid residues at positions 12, 13, 14, 15, 16, 17, 36, 37, 38, 39, 40, 61, 62, 63, 64, 65, or 67 (for SEQ ID NO: 4-28, residue numbering starting from the first amino acid after the optional N-terminal methionine residue; for SEQ ID NO: 29-30, residue numbering starting from the third amino acid (Cys residue) after the optional N-terminal methionine residue) are unchanged relative to the reference sequence. As described above, the amino acid residues at positions 12-17, 36-40, 61-65, and 67 of the polypeptide can directly contact avb6.

[0086] In another aspect, the polypeptide provided by the present invention has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to the amino acid sequences selected from SEQ ID NO: 4-30 and 36.

[0087] AEVRFVFRGDLTELMLRAVKDHLKKEGPHWNITSRGNELEVRGSHESDAKRIQKEFPSV QSTTQA (SEQ ID NO: 36).

[0088] In one embodiment, the polypeptide of this aspect has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an amino acid sequence selected from SEQ ID NO:21(E13T), 25(A39KG64R), and 29-30.

[0089] In one embodiment, residues 8-10 of SEQ ID NO: 4-30 (residue numbering starting from the first amino acid after the optional N-terminal methionine residue) or residues 10-12 of SEQ ID NO: 29-30 are unchanged. As described above, the interface residues for avb6 binding include residues 8-10 (or residues 10-12 in SEQ ID NO: 29-30). In another embodiment, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, sixteen, sixteen, or seventeen amino acid residues at positions 12, 13, 14, 15, 16, 17, 36, 37, 38, 39, 40, 61, 62, 63, 64, 65, or 67 are all unchanged relative to the reference sequence. For SEQ ID NO: 4-28, residue numbering begins from the first amino acid after the optional N-terminal methionine residue; for SEQ ID NO: 29-30, residue numbering begins from the third amino acid (Cys residue) after the optional N-terminal methionine residue. As described above, amino acid residues at positions 12-17, 36-40, 61-65, and 67 of the polypeptide can directly contact avb6.

[0090] In one embodiment of all the above embodiments, the amino acid change from the reference protein can be a conserved amino acid substitution.

[0091] The term "conservative amino acid substitution" used here refers to:

[0092] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Sce, Sme, Val, Ile, Leu) can only be replaced by other hydrophobic amino acids;

[0093] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) can only be replaced by other hydrophobic amino acids with large side chains.

[0094] Amino acids with positively charged side chains (Arg, His, Lys) can only be replaced by other amino acids with positively charged side chains;

[0095] Amino acids with negatively charged side chains (Asp, Glu) can only be replaced by other amino acids with negatively charged side chains; and

[0096] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) can only be replaced by amino acids with other polar, uncharged side chains.

[0097] In one embodiment, the polypeptide of the present invention can be linked to a detectable tag. This embodiment can be used, for example, for diagnostic purposes of the polypeptide. Any suitable detectable tag can be considered suitable for the intended use, including but not limited to radiolabels, fluorescent or luminescent proteins, avidin, biotin, or enzymes such as peroxidase.

[0098] In all embodiments, the peptide binds to α(v)β(6) integrin (avb6), as demonstrated by biomembrane interferometry using a his-labeled Ni NTA sensor, as described in the examples below. In one embodiment, as described in the examples below (Table 5), the peptide binds to avb6 with sub-nanomolar binding affinity using biomembrane interferometry with a his-labeled Ni NTA sensor. In another embodiment, when stably transfected with K562 cells, the peptide binds to avb6 with at least 100-fold selectivity compared to the corresponding integrins α(v)β(8) integrin (avb8), α(v)β(1) integrin (avb1), α(v)β(3) integrin (avb3), α(v)β(5) integrin (avb5), α5β1 (a5b1), α8β1 (a8b1), and α(iib)β(3) integrin (aiibb3).

[0099] In another aspect, the present invention provides nucleic acids encoding polypeptides of any embodiment or combination thereof. The nucleic acid sequence may comprise single-stranded or double-stranded RNA or DNA in genomic or cDNA form, or a DNA-RNA hybrid, wherein each may comprise chemically or biochemically modified, non-natural, or derived nucleotide bases. Such nucleic acid sequences may include additional sequences for facilitating the expression and / or purification of the encoded polypeptide, including but not limited to polyA sequences, modified Kozak sequences, sequences encoding epitope tags, output signal sequences, secretion signal sequences, nuclear localization signal sequences, and plasma membrane localization signal sequences. It will be apparent to those skilled in the art, based on the teachings herein, what kind of nucleic acid sequence can encode the polypeptide of the present invention.

[0100] In another aspect, the present invention provides an expression vector comprising a nucleic acid of any aspect of the present invention, the nucleic acid being operatively linked to a suitable control sequence. An "expression vector" includes a vector operatively linking a nucleic acid coding region or gene to any control sequence capable of influencing the expression of a gene product. A "control sequence" operatively linked to a nucleic acid sequence of the present invention is a nucleic acid sequence capable of influencing the expression of a nucleic acid molecule. Control sequences do not need to be adjacent to the nucleic acid sequence, as long as they serve to guide its expression. Thus, for example, an incorporated untranslated but transcribed sequence may exist between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered "operatively linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type, including but not limited to plasmids and viral-based expression vectors. The control sequence used to drive the expression of the disclosed nucleic acid sequence in a mammalian system can be constitutive (driven by any of a variety of promoters including but not limited to CMV, SV40, RSV, actin, and EF) or inducible (driven by any of inducible promoters including but not limited to tetracycline, ecdysone, and steroid-reactive agents). The expression vector must be capable of replicating as a free gene within the host organism or integrating into the host's chromosomal DNA for replication. In various embodiments, the expression vector may include plasmids, viral vectors, or any other suitable expression vector.

[0101] In another aspect, the present invention provides host cells or recombinant cells comprising the nucleic acids, expression vectors (i.e., free or chromosomally integrated) and / or polypeptides disclosed herein, wherein the host cells may be prokaryotic or eukaryotic cells. Cells can be transiently or stably engineered to incorporate the expression vectors of the present invention using techniques including, but not limited to, bacterial transformation, calcium phosphate coprecipitation, electroporation or liposome-mediated, DEAE-dextran-mediated, polycation-mediated, or virus-mediated transfection.

[0102] In another aspect, the present invention provides a pharmaceutical composition comprising:

[0103] (a) Any polypeptide, nucleic acid, expression vector, or host cell of any embodiment or combination of embodiments disclosed herein; and

[0104] (b) A drug-acceptable carrier.

[0105] The pharmaceutical compositions of the present invention can be used, for example, in the methods of the present invention described herein. In addition to the polypeptides of the present invention, the pharmaceutical compositions may also contain (a) a lyophilization protectant; (b) a surfactant; (c) a swelling agent; (d) a permeation regulator; (e) a stabilizer; (f) a preservative and / or (g) a buffer solution.

[0106] In some embodiments, the buffer solution in the pharmaceutical composition is Tris buffer, histidine buffer, phosphate buffer, citrate buffer, or acetate buffer. The pharmaceutical composition may also include a lyophilization protectant, such as sucrose, sorbitol, or trehalose. In some embodiments, the pharmaceutical composition includes a preservative, such as benzalkonium chloride, phenethylamine, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a swelling agent, such as glycine. In other embodiments, the pharmaceutical composition includes surfactants such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan laurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, or combinations thereof. The pharmaceutical composition may also include osmotic modifiers, such as compounds that make the formulation substantially isotonic or isotonic with human blood. Exemplary osmotic modifiers include sucrose, sorbitol, glycine, methionine, mannitol, glucose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition further includes a stabilizer, such as a molecule that, when bound to a protein of interest, can substantially prevent or reduce the chemical and / or physical instability of the protein in its lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0107] The polypeptide, nucleic acid, expression vector and / or host cell may be the sole active agent in the pharmaceutical composition, or the composition may also contain one or more other active agents suitable for the intended use.

[0108] On the other hand, the present invention provides the use of any embodiment or combination of embodiments disclosed herein of peptides, nucleic acids, expression vectors, host cells or pharmaceutical compositions for any suitable purpose, including but not limited to treating and / or detecting avb6(+) tumors in vivo, blocking avb6-mediated TGF-B signaling in vitro, and treating pulmonary fibrosis such as idiopathic pulmonary fibrosis (IPF).

[0109] In another aspect, the present invention provides a method for treating avb6(+) tumors or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis (IPF)), comprising administering to a subject in need an amount of any embodiment or combination of embodiments disclosed herein of a polypeptide, nucleic acid, expression vector, host cell, and / or pharmaceutical composition to effectively treat the subject’s tumor or IPF.

[0110] As described in the embodiments, α v High levels of β6 expression are associated with poor overall survival in a variety of cancers, including non-small cell lung cancer (NSCLC) and pancreatic cancer. v β6-mediated TGF-β activation is also associated with a variety of fibrotic diseases and is an established target for interventional therapy in idiopathic pulmonary fibrosis (IPF). IPF is a rare (10-60 cases per 100,000 people) progressive fibrotic lung disease of unknown cause, currently incurable, and accounts for 57% of all lung transplants. Pulmonary fibrosis may occur in patients with acute respiratory distress syndrome (ARDS) due to the ongoing and worsening COVID-19 pandemic, particularly in high-risk elderly populations, where patchy ground-glass opacities in lung tissue are observed. Recent studies have shown that SARS-CoV-2 infection, along with other drivers of pulmonary fibrosis following severe lung injury, increases the expression of TGF-β mRNA in the lungs and lung tissues. Therefore, in one implementation scheme, the subjects are human subjects with IPF who are also infected with SARS-CoV-2.

[0111] Subjects may be any suitable subjects, including but not limited to human subjects. As used herein, “treatment” means achieving one or more of the following: (a) reducing the severity of the disease; (b) limiting or preventing the development of characteristic symptoms of the disease; (c) suppressing the exacerbation of characteristic symptoms of the disease; (d) limiting or preventing the recurrence of the disease in subjects who have previously had the disease; and / or (e) limiting or preventing the recurrence of symptoms in subjects who have previously experienced symptoms of the disease. Achieving any number of such “treatments” would be highly beneficial to subjects with AVB6(+) tumors or pulmonary fibrosis.

[0112] α v The short serum half-life (<2 hours) of β6-targeted binding, high specificity and affinity, ease of production with *E. coli*, superior thermal stability, and aerosol formulation of the peptide (as described in the examples below) represent a significant improvement over existing therapies. Compared to antibody inhibitors, the peptides of the present invention can be formulated for tissue-specific delivery with a built-in tunable serum half-life and reduced systemic exposure, both of which promise to improve safety and reduce unwanted side effects (e.g., aerosol α for IPF). vThe lung retention and short serum half-life of β6 adhesive therapy may support better outcomes in the final lung transplantation configuration for IPF patients.

[0113] The method may include administration by a visiting medical professional via any appropriate route deemed suitable, including but not limited to pulmonary delivery (including but not limited to inhalation and nebulization), intravenous delivery, and intramuscular delivery.

[0114] In another aspect, the present invention provides a method for detecting avb6(+) tumors, comprising administering to a subject suspected of having avb6(+) tumors a certain amount of a polypeptide, nucleic acid, expression vector, host cell and / or pharmaceutical composition of any embodiment or combination thereof disclosed herein that can effectively detect tumors in the subject.

[0115] In all implementation schemes, the subject can be any suitable subject, including but not limited to mammals, such as humans.

[0116] In another aspect, the present invention provides a method for designing avb6-binding peptides, including the steps of any embodiment or combination of embodiments disclosed herein. The following examples provide detailed information.

[0117] Example

[0118] Integrin α v β6 is an important therapeutic target, associated with the activation of TGF-β1 / β3, upregulated in various cancers, and a major driver of fibrotic diseases including idiopathic pulmonary fibrosis (IPF), which can be triggered by coronavirus-induced acute respiratory distress syndrome (ARDS). However, few highly specific inhibitors of avyb6 have been developed. We describe the de novo design of an ultrastable inhibitory protein that binds to human α-alpha with sub-nanomolar affinity. v The β6-linked integrin exhibits >2000-fold specificity compared to other RGD (Arg-Gly-Asp)-binding integrins. The inhibitor's crystal structure closely matches the designed model, and its affinity and specificity stem not only from the ring-containing RGD but also from the second ring contacting the β6 subunit. The designed inhibitor blocks α-... v β6-mediated TGF-β signaling, and the realization of α in vivo v Specific targeting of β6(+) tumors. When administered via intraperitoneal injection, the engineered inhibitor demonstrated significant therapeutic effects against bleomycin-induced IPF in mice and showed promising preliminary efficacy as an inhaled therapy. In summary, these results demonstrate the ability of de novo protein design to create highly specific integrin inhibitors with therapeutic potential in immuno-oncology and pulmonary fibrosis.

[0119] Foreword

[0120] During tumor cell migration, wound healing, and inflammation, tissue reprogramming occurs, α v Upregulated β6 expression. High levels of α6 expression are observed in various cancers, including non-small cell lung cancer (NSCLC) and pancreatic cancer. v β6 expression was associated with poor overall survival. α v β6-mediated TGF-β activation is also associated with various fibrotic diseases and is a known target for interventional therapy in idiopathic pulmonary fibrosis (IPF). IPF is a rare (10-60 cases per 100,000 people) progressive fibrotic lung disease of unknown cause, currently incurable, and accounts for 57% of all lung transplants. Patchy ground-glass opacities in the lung tissue of patients with acute respiratory distress syndrome (ARDS) due to the ongoing and worsening COVID-19 pandemic, particularly in high-risk elderly populations, may indicate pulmonary fibrosis. Recent studies have shown that SARS-CoV-2 infection and other drivers of pulmonary fibrosis following severe lung injury increase the expression of TGF-β mRNA in the lungs and lung tissues.

[0121] Based on α v The crystal structure of the complex of β6 with the RGD-containing peptide (pdb ID 4UM9), and similar to other structures of the RGD-containing peptide bound to integrin, shows that the arginine and aspartic side chains form multiple hydrogen bonds with residues at the interface between the integrin α and β subunits. At the C-terminus of the RGD, the peptide has an α-helical turn, with the two leucine residues at the turn fitting into a hydrophobic bag formed by the β6 subunit ring. We attempted to... v A peptide containing RGD in the β6 complex structure is bound to a de novo-designed protein possessing the properties required for a therapeutic candidate. We first screened candidate topologies on a silicon wafer to extend the peptide to an 8-residue inverted conformation (RGDLGALA (SEQ ID NO: 31, ...). Figure 1 a). We searched the PDB database for low RMSD matches to the peptide skeletal conformation and extracted fragments consisting of the matching peptide plus five N-terminal flanking residues and five C-terminal flanking residues. These extended fragments were then superimposed onto the binding peptide conformation in the complex structure, and fragments that conflicted with the integrin at the skeletal level were discarded. Figure 1 b).

[0122] We discovered small α / β ferroredoxin structures ( Figure 1 c) and d) a binding loop can be constructed without collision with integrins, and this fold can be selected for subsequent de novo design calculations. A two-step approach is used to design the α-fold of ferrugin. vβ6 binder: The first step is to assemble the structure from fragments according to the rules for building an ideal protein: sampling different α-helical, β-sheet, and loop lengths, while using Rosetta... TM The torsion angle of the region corresponding to the RGD peptide is restricted to the torsion angle observed in the co-crystal structure. Figure 1 c). In the second step, the idealized ferricyanide fold structure obtained is superimposed on the binding loop and then combined with α. v β6 integrin docking was performed, and the amino acids on the binding surface were optimized to achieve low-energy interactions with the target. During these design calculations, the binding RGD motif remained immobilized. Figure 1 d). Perform de novo structural prediction tests on the final design model to determine that the design structures of these sequences are in the lowest energy state. Figure 1 e). Unlike most de novo protein-protein interfaces designed previously, α v All design interactions between β6 and the designed microproteins are mediated by loops. Figure 1 f). Besides the RGD ring, there are two others related to α. v Other rings in contact with the β6 subunit: Ring 1 connects sheets 2 and 3 to the β6 subunit; Ring 2 connects helical 2 and sheet 4 to the α subunit. v Subunit contact ( Figure 1 f).

[0123] We obtained nine synthetic genes encoding designs of different lengths of helices, chains, and loops (combination details are shown in Supplementary Materials). In initial testing of candidate conjugates expressed on yeast cell surfaces, four designs bound fluorescently labeled α-carboxylates in the expected metal cation-dependent manner. v β6 (a biotinylated variant labeled with streptavidin, R-phycoerythrin conjugate (SAPE)) binding. Using the strongest binder design 2 as the starting template, we constructed and screened by error-prone PCR, and obtained a new variant with 5 mutations (02_E2V_T12A_E40V_S44I_T63I) after display on three rounds of yeast surface and fluorescence-activated cell sorting (FACS). We resolved the crystal structure of the variant (SEQ ID NO: 36) at 2A resolution. While a portion of the crystal structure (including the RGD ring) overlapped well with the computationally designed model, the last helix of the fold was rotated half a turn. This is likely driven by the partially exposed Phe56 in the initial design model. Figure 4 ).

[0124] In order to generate α that has broader contact with integrins v In the second design round, we connected the crystal structure of the β6 conjugate from the first design round to the α conjugate by superimposing RGD rings. vOn β6, two loop regions close to the integrin were identified in the design. We sampled a series of loop lengths and conformations for the two loops and selected 16 designs predicted to have specific interactions with the integrin for experimental testing. We obtained synthetic genes encoding these 16 designs and used yeast surface display and SAPE-tagged biotinylated α-coated β6 loops for experimental testing. v The binding of β6 protein was measured. Of the 16 ordered designs, 12 were well expressed on the yeast surface and were found to bind α in a metal-dependent manner. v β6(Ca(II), Mg(II), Figure 5 (FACS data). We used four different concentrations of biotinylated α. v β6 (50 pM, 100 pM, 300 pM, and 500 pM, supplementary information) was used to determine the binding on the yeast surface. Based on yeast surface display experiments (… Figure 6 ), αv6_3 shows the α v β6 showed the strongest binding signal and bound more tightly than the original crystalline variant. We expressed and purified the five strongest binding compounds (av6_3, av6_7, av6_9, av6_11, and av6_15) in *E. coli*. We also identified av6_3 as a counterpart to another integrin α responsible for TGF-β activation. v β8, for α v β6 has the highest selectivity ( Figure 7 In αv6_3, the typical RGDLXXL (SEQ ID NO: 32) motif of the TGF-β1 precursor domain is incorporated into a loop that contacts the sheet 1 and helix 1 of the ferrenoprotein fold. Figure 1 g). The amphiphilic helix behind the RGD ring is closely attached to the hydrophobic groove formed by the β6 subunit. Figure 1 h), mimicking TGF-β1 peptide and α v The binding effect of β6. Asn37 forms a hydrogen bond with Asp901 from the β6 subunit, and connects with α. v The main chain atoms of β6 residues Ser756 and Ile757 form Arg61 hydrogen bonds. Figure 1 i). All residue numbers are based on Rosetta. TM The internal numbering system starts from the first residue of the design compound.

[0125] To explore the sequence determinants of binding, each residue in av6_3 was mutated to one of the other 19 amino acids, and α was also modified. v β6 binding was used for two rounds of yeast surface display and FACS sorting (FACS, Figure 8 Deep sequencing of the library before and after selection identified the substitutes enriched after binding selection (see Tables 1-3). Dominant α vThe core residues of the β6 microconjugate are largely conserved, suggesting that the designed residues are close to optimal folding. Figure 2 a) Due to the importance of this tripeptide motif pair binding, mutations in the RGD loop were, as expected, highly absent. Five mutations were enriched at the interface: two mutations (E13 / A39) interacting with the β6 subunit, and one mutation, M15, located at the α... v Between the groove formed by the β6 subunit and the α subunit, P63 / G64 and α v Subunit interactions. Following the amphiphilic helix, E13 is more inclined to hydrophobic or small polar residues, likely due to the negatively charged proximal residues on the integrin; threonine enriched at this position is also present in the TGF-β1 peptide. Most other highly enriched alternatives also involve charge complementarity: M15R / K at α v Within the range of hydrogen bond interactions between D220 and Y250 of β6 ( Figure 2 b). Ring 2 facing α v The two consecutive residues (P63 and G64) of the subunit are enriched with positively charged Lys or Arg residues, which may interact with the receptor α. v The two acidic residues D218 and D220 on the subunit form a salt bridge. Figure 2 c and 2d). The A39K substitution facing the β6 subunit may introduce a salt bridge with Glu963 ( Figure 2 e). We expressed, purified, and tested a total of nine mutants (alone and in combination) of these selected alternatives, and used biolayer interference (BLI) measurements. All variants were α-resistant. v β6 exhibits sub-nanomolar binding affinity, while the original uninvolved av6_3 binds to α. v β6 binding, Kd = 1.18 nM (see Table 5). Two high-affinity variants were selected for further characterization: BP1 (av6_3_E13T) and BP2 (av6_3_A39KG64R), the former having a single substitution that more tightly reproduces the TGF-β1 peptide sequence, and the latter having two substitutions that introduce a positive charge, similar to α... v The negative charges in the two subunits of β6 integrin are complementary.

[0126] Table 5

[0127]

[0128]

[0129] Functional characteristics of the designed inhibitor:

[0130] TGF-β is an inactive complex formed with latent-associated peptide (LAP); α vβ6 binds to this inactive LAP (TGF-β complex) and releases active TGF-β. This active TGF-β then interacts with TGF-βRI / RII and triggers downstream signaling. α v β6 expression is primarily confined to epithelial cells. Under normal physiological conditions, it is mainly limited to tissues undergoing morphological changes during development, and is almost absent in fully differentiated epithelial cells. However, under pathological conditions, α... v β6 expression is upregulated during tissue reprogramming in tumor cell migration, wound healing, and inflammation, while α... v High levels of β6 expression are associated with poor overall survival in a variety of cancers, including non-small cell lung cancer (NSCLC) and pancreatic cancer. This has also led to research into targeting α6 in tumor immunotherapy. v Great interest in β6.

[0131] We began testing these conjugates with α v The ability of β6(+) cells to bind and block TGF-β-mediated downstream signaling. We generated fluorescently labeled BP1 and BP2 by binding Alexafluor-488 to engineered C-terminal cysteine ​​residues via maleimide chemistry. The fluorescently labeled proteins were then used to target α... v β6-positive human epidermoid carcinoma A431 cells were titrated. BP1 and BP2 were bound to A431 cells with Kd values ​​of 167 (±.028) pM and 30 (±.004) pM, respectively (data not shown).

[0132] Next, we investigated the ability of these designed conjugates to block TGF-β signaling using transformed mink lung reporter cells (TMLCs), which produce luciferase in response to active TGF-β. Both BP1 and BP2 blocked α v β6-mediated TGF-β activation, IC 50 The values ​​were 199 pM (95% CI [119 pM, 332 pM]) and 151 pM (95% CI [79.6 pM, 284 pM]), respectively. Figure 3 BP1 also blocks α. v β8-mediated TGF-β activation, while BP2 had no effect at the highest experimental concentration (333 ng / ml), consistent with their in vitro binding characteristics.

[0133] Because BP2 binds to A431 cells with a higher affinity than BP1, and also to α v β6 is more specific, so we chose BP2 for further in vivo experiments. To investigate whether evolved variants can interact with α in vivo... vβ6(+) tumor binding was chemically conjugated to Alexafluor-680C-2 maleimide, generating fluorescently labeled BP2 (AF680-BP2) via engineered C-terminal cysteine ​​residue. A431 cells (α-) were injected into the left and right shoulders of 6-8 week old female athymic nude mice. v β6(+)) and HEK 293T(α) v β6(-)). When the tumor diameter reached 5-10 mm, mice were injected with 1.5 nmol of AF680-BP2 protein. AF680-BP2 in α v Rapid accumulation occurred in β6-positive tumors, achieving good tumor-muscle fluorescence contrast within 3 hours post-injection (data not shown). In α... v No fluorescence was detected on β6-negative HEK-293T tumors, indicating that Alexafluor-680C-2 is in vivo effective against α-negative tumors. v β6 is selective. We also performed a semi-quantitative in vitro and in vivo biodistribution analysis of AF680-BP2. Fluorescence intensity analysis in different tissues showed that AF680-BP2 was selective in α-... v Accumulation was observed in β6-positive tumors and kidneys (tumor-to-kidney ratio 1:1.04, data not shown), with no significant off-target binding (including with α). v (β6-negative tumor binding). These results clearly demonstrate the selective targeting of α in vivo using designed conjugates. v β6-positive tumors. Furthermore, quantification of whole-body imaging data of AF680-BP2 after tail vein injection indicates that its serum half-life is approximately <2 hours (data not shown), and it can be filtered by the glomeruli, enters the urine through the kidneys, and is metabolized by the liver.

[0134] The specificity of the designed conjugate for other RGD-binding integrins is determined by:

[0135] As mentioned above, integrin α v β8 also plays a role in the activation of TGF-β1 / TGF-β3. α v β8 is overexpressed on T-reg cells and is crucial for suppressing T cell-mediated inflammation. For therapeutic applications, inhibition of α8 is more effective than overall inhibition of TGF-β. v β6-mediated TGF-β inhibition is desirable. In BP2, loop 2 is localized to confer specificity between the two integrins. Figure 2 j): K39 in ring 2 faces E963 in the β6 subunit and K901 in β8 ( Figure 2 j). Unlike α v β8,BP2 is used to stably express α v β6 / α vIn a cell surface binding assay of β8-containing K562 cells, the α-type... v The specificity of β6 is greater than 5000 times. BP1 with alanine (A39) at this position has much lower specificity. Figure 2 k). BP1 and BP2 do not cross-react with other RGD-binding integrins, including α. v β1, α v β3, α v β5, α5β1, α8β1 and α iib β3 (cell surface binding assay using K562 cells stably transfected with different RGD-binding integrins, at a concentration as high as 200 nM).

[0136] To further improve protein stability, we used Rosetta TM Paired sites were scanned to introduce disulfide bonds with optimal geometry, and four variants (two for each construct) were selected for experimental characterization. Both versions of the protein, with and without disulfide bonds, eluted as monodisperse peaks by volume exclusion chromatography. Designed circular dichroism (CD) spectra revealed two minima centered at 208 and 222 nm, consistent with mixed α / β folding (data not shown). The introduction of disulfide bonds made both proteins highly thermostable; based on CD spectral data under non-reducing conditions (data not shown), they maintained their secondary structure at 95 °C. We selected two of these proteins (BP1_disulf and BP2_disulf) for further in vitro and in vivo characterization. Both BP1_disulf and BP2_disulf could be purified in one step from E. coli cell lysates by boiling at 85 °C for 10 min. Figure 9 BP2_disulf binds to α with sub-nanomolar affinity. v β6 binding, the RGD to KGE knockout mutation cancels its binding with the receptor, confirming that the RGD loop is necessary for binding.

[0137] We resolved the crystal structure of the disulfide-stabilized version of BP1, which exhibits sub-nanomolar affinity with α... v β6-binding, and BP1_disulf does not melt at 95°C under XXARMSD. The crystal structure matches the design model very well, with a root mean square deviation (rmsd) of [value missing]. ( Figure 2 h). In the crystal structure, the disulfide bond also adopts a conformation similar to that of the design model described above. Figure 2 i) Most core hydrophobic residues employ the same rotatiforms as the design model. Unlike most designed protein inhibitors, most interactions in BP1_sulf are ring-mediated. There are three rings with α vβ6 contact: 1) RGD ring 2) Ring 1 and 3) Ring 2 ( Figure 2 f). The designed RGD binding loop is 5 residues long and uses a skeletal structure almost identical to the design model. Figure 2 j). Except for arginine, all residues in the circuit are rotate isomers similar to those in the design model. Figure 2 j). Following this is the RGD ring, and the LATL motif forms an amphiphilic helix that can closely attach to the hydrophobic groove on the β6 subunit. Previous studies have shown that α v β6 recognizes not only the RGD ring but also the amphiphilic helix formed by the LXXL (SEQ ID NO: 33) motif, which interacts only with β6 and provides a blueprint for ligand binding specificity and recognition beyond the RGD sequence. Ring 1 connects sheets 2 and 3 to contact the β6 subunit, which is designed to be GGGA-abego type. Ring 2 connects helix 2 and sheet 4 to contact the α subunit, which is designed to be BAAB-abego type. v Subi ( Figure 1 f) Contact. In the crystal structure, these two rings adopt a main chain conformation almost identical to the design model ( Figure 2 h). In the design model of the complex, Asn37 on ring 1 forms a hydrogen bond with Asp901 of the β6 subunit of the receptor, and with α. v In β6, the Ca(II) atoms are within the coordination distance, while α v β8 does not have this coordination distance. In the designed structure, the ideal positioning of ring 1 imparts specificity to the β subunit, providing an easy pathway to designing inhibitors with integrin subtype specificity.

[0138] BP2-disulf reduced the fibrotic burden and restored lung function in bleomycin-induced mice.

[0139] IPF is a progressive disease characterized by the formation of scar tissue in the lungs, with a median survival of 3 to 5 years after diagnosis. Patients experience progressive shortness of breath and impaired lung function, manifested as decreased forced vital capacity (FVC), reduced diffusion, and decreased oxygenation, eventually leading to respiratory failure. The progression of pulmonary fibrosis is partly due to alpha-12 pulmonary fibrosis. v β6 integrin activates TGF-β, worsening the Smad 2 / 3 pathway. TMLC assays confirmed that BP2 can specifically block α... v Following β6-mediated TGF-β signaling, we investigated the therapeutic effect of this molecule on bleomycin-induced pulmonary fibrosis in mice.

[0140] Twelve-week-old male C57BL / 6 mice were intratracheally injected with 50 μL of bleomycin (1 mg / kg body weight). From day 7 to day 19 following bleomycin infusion, mice were intraperitoneally injected with a BP2_disulf conjugate every other day for a total of seven treatments, compared to untreated mice (data not shown). Compared to the NY group, the bleomycin (BLM, bleomycin, and BP_2disulf treatment) group experienced weight loss (approximately 5-8% of initial body weight) (data not shown). However, BP2_disulf treatment reduced overall weight loss at 14 days post-lung injury compared to the BLM group (data not shown). High-resolution lung scans of the mice were obtained using a miniature CT scanner, allowing real-time visualization of fibrosis development. Lung injury was first detected on day 7 and became apparent on days 14 and 21 (data not shown). With BP2_disulf treatment, by day 21, the large fibrotic lesions prevalent in BLM mice were not observed in the lung tissue (data not shown). Mice treated with BP2_disulf did indeed suffer damage from bleomycin challenge, but the damage was not as severe as in the BLM group. Lung morphology in BP2_disulf-treated mice showed fibrotic lesions, but maintained alveolar spaces absent in bleomycin-challenged mice (data not shown). Untreated mice exhibited a fibrotic burden of 4.107%, bleomycin-challenged mice 11.01%, and BP2_disulf-treated mice 6.857% (data not shown). The fibrotic burden was significantly reduced in BP2_disulf and NT mice compared to the BLM group. Tissue density frequencies were collected by dividing Hounsefield unit intensities into several units and sampling the scanned images to obtain the frequency of unit intensities. The tissue density distribution shows a rightward shift in scanned images of bleomycin-damaged mice, indicating an increase in dense tissue compared to the NT and BP2_disulf-treated groups. The distribution in the BP2_disulf-treated group was comparable to that in the NT group (data not shown).

[0141] To confirm that BP2_disulf not only reduced bleomycin-induced fibrotic burden compared to the NT group, but also improved overall lung function, FlexiVent was used 21 days after bleomycin administration. TMRespiratory mechanics was measured using the FX system. Static compliance was measured as the elastic properties of the lungs and calculated based on the pressure-volume loop. BP2_disulf treatment induced a significant increase in static compliance in mice treated with bleomycin alone (data not shown), with similar elastic properties to untreated mice. Forced vital capacity (FVC) in BP2_disulf-treated mice showed similar airflow to NT mice, and were statistically significantly increased compared to BLM mice (data not shown). BP2_disulf treatment rescued bleomycin-induced IPF-like restrictive lung disease-induced lung function loss, with mean PV loop calculations showing little difference compared to NT mice and a 100% improvement compared to bleomycin-treated mice (data not shown).

[0142] discuss

[0143] The designed inhibitor (BP2_disulf) binds to α with high affinity and specificity. v β6 binding. The protein contains a single disulfide bond, is ultrathermally resistant, readily and efficiently expressed in *E. coli*, and can be purified from crude cell lysate in a single step by heating at 85°C. BP2_disulf was highly effective in a bleomycin-induced IPF mouse model (100 μg / kg). Mice treated with intraperitoneal injection every other day showed improvements in lung mechanics and histopathological changes. As an inhaled treatment of a bleomycin-induced IPF model, the protein showed the desired results (Supplementary Information). Due to the protein's high thermostability, it can also be formulated for nebulization. BP2_disulf retains its secondary structure after nebulization. This is particularly advantageous because the tissue-specific exposure of the conjugate is limited compared to the overall inhibition of TGF-β upon IP injection. The short serum half-life (<2 hours) of our designed micro-binding protein, and its α-binding... v The high specificity and affinity of β6-targeting, ease of production in *E. coli*, superior thermal stability, and aerosol formulation capabilities provide an improved spectrum of targeted products for novel therapeutic candidates in IPF. Compared to antibody inhibitors, the α-targeting inhibitors disclosed in this paper... v β6 conjugates can be formulated into tissue-specific delivery systems with built-in adjustable serum half-lives and reduced systemic exposure, both of which are expected to improve safety and reduce unwanted side effects (e.g., aerosol α for IPF). vThe short lung retention and serum half-life of β6 conjugate therapy could support better outcomes in the eventual lung transplantation scenario for IPF patients. The recent SARS-CoV-2 outbreak has also posed a serious threat to the lung health of older adults. While most affected individuals recover without major complications, ARDS patients experience severe lung damage / lesions and are expected to develop pulmonary fibrosis over time, similar to the previous SARS outbreak. Therefore, the de novo-designed protein reported in this article has considerable therapeutic potential for treating IPF, progressive respiratory diseases associated with current and future coronavirus infections, and cancer immunotherapy.

[0144] A common challenge in drug development is targeting a member of a large, closely related protein family. This can be difficult to achieve with small molecules, and developing an antibody panel capable of such differentiation is challenging because it may require a large number of negative selections. Our structure-based de novo design strategy provides a systematic approach to achieving this specificity by integrating previously known binding motifs and novel interactions into an ultrastable scaffold, thereby enabling targeting at α- v β6 and α v β8 provides higher affinity and specificity. A significant advantage of computational design compared to previous methods is the ability to combine RGD rings with additional rings containing K39, which imparts specificity, to form a minimally stable scaffold.

[0145] Materials and methods:

[0146] Computational techniques: The main text discusses the procedures for design schemes.

[0147] Yeast display: Standard yeast surface display techniques were used to screen for binding and directed evolution designs. Genes encoding these designs were cloned into a frame containing petcon2 with N-terminal aga2 and C-terminal myc tags. Surface expression of Myc was detected using an anti-C-Myc antibody and biotinylated human α-amino acids. v β6 binding was detected, and FACS staining was performed using streptavidin bound to phycoerythrin. Two different buffers were used for the binding and washing steps of yeast display: binding buffer 20 mM TRIS, 150 mM NaCl, pH 8.0, 1% BSA, 1 mM Ca(II) and 1 mM Mg(II), and washing buffer 20 mM TRIS, 150 mM NaCl, pH 8.0, 0.5% BSA, 1 mM Ca(II) and 1 mM Mg(II).

[0148] The SSM library was generated according to the previously described protocol by using mutant primers (sequences below) at each position. The resulting library was transformed into yeast via repeat electroporation (biological repeats). Sorting was performed in two rounds: first, the library was treated with 4 uM trypsin and 0.8 uM chymotrypsin for 5 minutes, followed by treatment with 200 pM biotinylated α-lysate. v β6 was labeled, and the first 5% of the conjugates were collected. In the second and final rounds of selection, 100 pM of biotinylated α-carboxylate was used. v β6 and rejection rate were used for selection. For the rejection rate selection step, 500 nM of untreated purified av6_3 was added to the cells, and the cells were tumbled at 37°C for 1 hour, then the top 1% of the binding population were selected. Figure 8 DNA was extracted from the library before and after classification and barcoded. Enrichment rates were calculated after sequencing the library using Illumina.

[0149] Protein Expression and Purification: Genes encoding protein variants were arranged as gblock gene fragments from IDT and cloned in pet29b between NdeI / XhoI restriction sites with C-terminal histidine residues. All mutant variants of these proteins were expressed in BL21(DE3*) using Studier self-induction technology in standard shake flasks at 25°C for 36 h. Cells were collected and resuspended in 20 mM Tris, 250 mM NaCl, and 20 mM imidazole (lysis buffer). Cells were lysed using a microfluidic apparatus and cell debris was separated by centrifugation at 24000 g for 45 min. Soluble proteins were first purified using a standard Ni-NTA affinity column, followed by volume exclusion chromatography (S7510 / 300 upscaling) on ​​a GE-Akta purification FPLC system. Peaks corresponding to the monomeric proteins were collected and further validated by mass spectrometry. For the bleomycin-induced IPF model, the protein was further purified to achieve endotoxin levels <5 EU / ml.

[0150] Biotinylation of the designed protein: To generate single-biotinylated proteins, an avi tag sequence (GLNDIFEAQKIEWHE; SEQ ID NO: 34) was introduced at the N-terminus of the protein. The protein was biotinylated by co-conversion of the protein of interest with pBirA (a vector encoding an E. coli biotin ligase for in vivo biotinylation); or by using purified protein and an in vitro biotinylation kit, employing the manufacturer's protocol to form affinity. Biotinylation was further confirmed by mass spectrometry.

[0151] Structural analysis of designed proteins :

[0152] To determine the crystal structure of BP1_disulf, we expressed BP1_disulf using an Nterm-TEV-cleavable HIS tag. After protein expression and purification, BP1_disulf was treated with a (1 / 100) diluted storage TEV protease and dialyzed against TBS overnight at room temperature. Following cleavage as monitored by SDS-page gel electrophoresis, the protein was run on a second-gravity Ni-NTA column to separate the cleaved his tag and his-tagged TEV from the cleaved protein. After his tag cleavage, the protein was concentrated to approximately 50 mg / mL and subjected to a crystallization assay. The bound protein and BP1_disulf were crystallized by vapor diffusion at 24 °C by mixing with equal volumes of storage solutions: 0.2 M KNO3, 20% PEG3350 and 0.2 M K3Citrate, 20% PEG3350, respectively. The crystals were briefly freeze-soaked in a storage solution containing 15% PEG200 and then rapidly frozen in liquid nitrogen. Diffraction data were collected using a MAR225 CCD detector on a GM / CA beamline at an Advanced Photon Source (APS) at -173°C and processed using XDS. Interestingly, the diffraction data of the binding protein, initially scaled to space group P6122, exhibited large Patterson peaks at 1 / 3 and 2 / 3 of the c-axis, indicating two translated NCS molecules along the c-axis. A solution was found using a designed model. The model was tested at Rosetta... TM The data was corrected and then reconstructed using phenix.autobuild. Autobuild was able to reconstruct most of the sequence of the model, but R and Rfree were still very high, with good electron density maps at the 44% / 47% level. The data was then rescaled to space group P31, the structure was corrected, and each asymmetric unit contained 12 molecules with tetrahedral twinning. AUTOBUILD TM Used to construct one-third of the sequence, and manually constructed and used in COOT with Phenix. TM and RefMAC TM It is used multiple times in the first few steps of many refined iterations. MolProbity TM Used to verify the final structure.

[0153] Table 6. Statistics and Structure Corrections for X-ray Diffraction

[0154]

[0155]

[0156] The number in parentheses indicates the highest resolution shell.

[0157] b Rmerge=Sh Si|Ii(h)-<I(h)> | / ShSi Ii(h), where Ii(h) and<I(h)> is i th This means the measurement of the reflection intensity h.

[0158] c Each unique reflection 28 The Pearson correlation coefficient between the average strengths of the random semi-datasets.

[0159] d Sh||Fobs(h)|-|Fcalc(h)|| / Sh|Fobs(h)|, where Fobs(h) and Fcalc(h) are the observed and calculated structure coefficients, respectively. No I / s(I) has been applied.

[0160] e using Molprobity 18 calculate.

[0161] Biophysical characterization of the designed protein :

[0162] Secondary structure and thermal stability of proteins were measured using a JASCO-1500CD instrument. For normal wavelength scans, 10–15 μM of protein in TBS (20 mM TRIS, 50 mM NaCl, pH 8.0) was used. The CD spectroscopy measurement range was 240–195 nm, with a scan rate of 100 nm / min. For the thermal melting experiment, the signal intensity at 222 nm was monitored as a function of temperature (4 °C–95 °C) at a temperature gradient of 2 °C / min. Samples were held at the specified temperature for at least 5 seconds before measurement. To investigate the effect of engineered disulfide bonds on stability, 1 mm TCEP was added to the protein to measure thermal stability under reducing conditions.

[0163] Biological layer interference for determining protein binding kinetics :

[0164] Data collection at Octet TM On a RED96 (Forte Bio) sensor, the protein conjugates were processed using instrument software. His-tagged protein conjugates were immobilized on a Ni-NTAoctet sensor. The tip was then immersed in a solution containing different concentrations of α... v In the β6 wells. Binding and dissociation steps were recorded at 900 sec and 1200 sec, respectively. An empty sensor without loaded binding protein was included to discard any nonspecific binding of αvβ6 to the octet tip.

[0165] fluorescent tags of the designed conjugate :

[0166] For in vitro binding assays and in vivo imaging assays, the designed conjugates were used with Alexa Fluor, respectively. TM488C5 maleimide and Alexa Fluor TM 680C2 maleimide (Thermo Fisher Scientific) is used for labeling via a single C-terminal cysteine ​​variant. In a typical labeling experiment, 50–200 μm of protein is reduced with 1 mM TCEP for 30 min at room temperature. A 3–5 mol excess of maleimide is added to the protein solution and the mixture is incubated overnight at room temperature. The reaction mixture is then purified on an S75-10 / 300 column to separate the free dye from the labeled protein. Fluorescent coupling is further confirmed by mass spectrometry.

[0167] In vitro binding assay using fluorescently labeled conjugates :

[0168] Epidermal carcinoma cells (A431) and human embryonic kidney 293T cells (HEK 293T) were purchased from the American Type Culture Collection (ATCC) and grown in Dulbecco modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37°C in humidified air with 5% CO2. Binding assays were performed on A431 cancer cells. A431 cells were isolated from the culture flasks using enzyme-free cell dissociation buffer (Gibco). Different concentrations of AG-AF488 and E13T-AF488 were mixed with 5 x 10⁻⁶ cells / mL of AG-AF488. 4 A431 cells were cultured in a solution containing 0.1% BSA and 1 mM Ca. 2+ and 1mM Mg 2+ Incubate (BTB) in 1X TBS, suspend and rotate at 4°C for 5 hours. Use sufficient incubation volume to avoid ligand consumption exceeding 5%. After culture, wash cells with BTBS and run on Accuri TM Analysis was performed using flow cytometry on the C6 instrument (BD Biosciences) and using FlowJo... TM The software (TreeStar) quantifies the data. The Kd value is determined using Prism. TM 7 (GraphPad Software) uses data to fit a site-specific binding curve to determine the binding.

[0169] Using TMLC analysis, α was inhibited by a designed inhibitor. v β6-mediated TGF-β activation:

[0170] Table 7 provides the commercial sources and descriptions of the reagents used in TMLC analysis.

[0171] Table 7

[0172]

[0173]

[0174] Recombinant human TGFb1 (R&D system, catalog 240-B-010)

[0175] According to the manufacturer's instructions, the luciferase detection system (Promega, catalog E1501) uses Reporter lysis buffer 5X: 5X (Promega E397A).

[0176] 96-well cell culture plate (Costar catalog 7107)

[0177] 96-hole white-bottomed white-walled polystyrene Optiplates TM (Perkin Elmer 6005290)

[0178] Antibody

[0179] Anti-TGFb1,2,3mIgG1 clone 1D11 (R&D MAB1835-500) 0.5mg / ml

[0180] Mouse IgG1 isotype control clone 11711 (R&D MAB002) 0.5 mg / ml

[0181] Anti-av (CD51) mIgG1 clone L230 (EnzoALX-803-304-C100) 0.1 mg / ml; Anti-avb63G9 (internal) hIgG1 SP16-1061 0.21 mg / ml

[0182] NIP228 hIgG1 (3G9 isotype) (internal)

[0183] Anti-AVB8 (internal)

[0184] NIP228 hIgG1 (anti-avb8 isotype) (internal)

[0185] Detailed protocol for TMLC detection of anti-AVB6 / B8264RAD (internal) BPD.95SP10-36210.45mg / ml:

[0186] Co-culture detection device

[0187] Detection medium: DMEM + 1% FBS + Pen / strep

[0188] 1. Use acutase to remove 1 LF of TMLC cells from the flask.

[0189] Wash in 10 ml PBS and add 5 ml accutase / flask.

[0190] Incubate at 37°C for 3-5 minutes.

[0191] Add the test culture medium and rotate at 300x g for 5 minutes.

[0192] Resuspend and count in 5 ml of test culture medium.

[0193] Suspended in 55 ml of detection medium, 3.3e6 / ml, total 1.65e7.

[0194] 2. After counting using the trypan blue exclusion method, the cells were suspended in analytical medium with a concentration of 300,000 cells / ml.

[0195] 3. Add 50 μL of cell suspension (15,000 cells / well) to each well of a 96-well tissue culture plate (see plate layout).

[0196] 4. Place the cells for 3 hours to allow TMLC adhesion.

[0197] 5. Prepare ABS and binding proteins at twice the final concentration.

[0198] 6. Add 200 μl of abs, binding protein, or culture medium to the appropriate wells in a 96-well PP plate.

[0199] 7. Prepare a 2 μg / ml rhTGF-b1 detection medium.

[0200] The stock volume is 20 μg / ml: 10000-fold dilution (1 / 100, then 1 / 100).

[0201] 2 μl + 198 μl of culture medium

[0202] 15 μl (1 / 100) + 1485 μl culture medium.

[0203] Prepare cells at a concentration of 2x.

[0204] 1) HeLab8 cells were removed from the flask using accutase. After counting using trypan blue exclusion, the cells were suspended at 300,000 cells / ml.

[0205] In 42.1 ml, 2.52e6 / ml, total suspension 1.263e7.

[0206] 2) Take approximately a number of K562 parental cells and avb6 transfected cells (30 ml), and rotate at 300 x g for 5 minutes. Resuspend the cell particles in 5 ml of assay medium and count them. (The text abruptly ends here, so the translation stops as well.) 6 Cells were suspended at a concentration of / ml.

[0207] K562 parental cells were suspended in 17.7 ml, 4.25e6 / ml, for a total of 2.125e7.

[0208] K562avb6 was suspended in 11.4 ml, 2.725e6 / ml, totaling 1.36e7.

[0209] 3) Add 200 μl of cells or culture medium or 2x rhTGF-b1 to the appropriate well in a deep-well PP plate containing the binding protein, antibody or culture medium (see step 6).

[0210] 4) Incubate at room temperature for 15 minutes to allow the binding protein / antibody to bind to the cells (TGF-b).

[0211] 5) Aspirate the culture medium and add 100 μl of cell + / - Abs, culture medium, or 1 ng / ml rhTGF-b1 + / - Abs to the appropriate wells (see plate layout).

[0212] 6) Before measuring luciferase activity, culture the cells at 37°C and 5% CO2 for 18-20 hours.

[0213] Luciferase assay

[0214] 1. Use a multichannel method to remove 70 μL of culture supernatant and store it in a 96-well U-bottom polypropylene plate at -80°C for future detection of cytokines / matrix metalloproteinases.

[0215] 2. Wash the cells twice with 200 μL / well PBS (absorb the cells between washes). Losing K562 cells will not affect the outcome.

[0216] 3. Add 100 μl of 1×Reporter lysis buffer (1 part 5x lysis buffer + 4 parts distilled water) to each well and freeze-thaw the cells in a -80°C freezer to fully lyse the cells.

[0217] 4. Prepare luciferase detection buffer by thawing it to room temperature and then adding it to the lyophilized luciferase detection substrate.

[0218] 5. Transfer 80 μl of cell lysate to a white transparent plate and add 100 μl of luciferase assay reagent.

[0219] 6. Immediately read the signal on the photometer.

[0220] Envision Ultrasensitive Emission Analysis (96)

[0221] Statistical analysis

[0222] All values ​​are reported as mean ± standard deviation. Data analysis was performed using one-way ANOVA followed by multiple comparisons using Tukey post-hoc tests. GraphPad was used. TM Analysis and plotting were performed using Prism 6.0 (GraphPad, San Diego, USA). Results with a p-value < 0.05 were considered statistically significant.

[0223] The sequences of all designed and evolved variants reported in this paper are shown in Table 4.

[0224] The description of embodiments of the invention is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Specific embodiments and examples of the invention are described herein for illustrative purposes, but as those skilled in the art will recognize, various equivalent modifications can be made within the scope of the invention. sequence list <110> University of Washington <120> Computational design of α(v)β(6) integrin-binding proteins <130> 19-1733-PCT (48279.02WO2) <150> US 62 / 925,868 <151> 2019-10-25 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 72 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Amino acid X can be A, C, D, E, F, G, I, K, L, M, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (2)..(2) <223> Amino acid X can be V, A, C, D, E, G, I, L, M, R, S, T, W, or Y. <220> <221> MISC_FEATURE <222> (3)..(3) <223> Amino acid X can be V, A, C, D, F, G, I, L, N, or Q. <220> <221> MISC_FEATURE <222> (4)..(4) <223> The amino acid X can be R, A, C, F, G, I, K, M, N, S, or V. <220> <221> MISC_FEATURE <222> (5)..(5) <223> Amino acid X can be F, A, C, E, G, H, I, K, L, M, N, P, R, V, W, or Y. <220> <221> MISC_FEATURE <222> (6)..(6) <223> The amino acid X can be V, A, D, G, H, I, K, L, M, N, Q, R, S, or T. <220> <221> MISC_FEATURE <222> (7)..(7) <223> Amino acid X can be F, A, C, D, G, H, I, K, L, R, S, T, V, or Y. <220> <221> MISC_FEATURE <222> (8)..(8) <223> The amino acid X can be R, E, G, I, K, S, T, or V. <220> <221> MISC_FEATURE <222> (9)..(9) <223> The amino acid X can be G, C, D, R, or S. <220> <221> MISC_FEATURE <222> (10)..(10) <223> Amino acid X can be D, A, E, H, V, or Y. <220> <221> MISC_FEATURE <222> (11)..(11) <223> The amino acid X can be L, F, M, Q, or S. <220> <221> MISC_FEATURE <222> (12)..(12) <223> The amino acid X can be A, E, G, K, P, R, S, T, or V. <220> <221> MISC_FEATURE <222> (13)..(13) <223> Amino acid X can be E, A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (14)..(14) <223> The amino acid X can be L, E, F, K, M, R, S, or V. <220> <221> MISC_FEATURE <222> (15)..(15) <223> The amino acid X can be M, A, C, E, G, H, I, K, L, N, P, Q, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (16)..(16) <223> The amino acid X can be L, A, F, G, K, M, N, Q, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (17)..(17) <223> The amino acid X can be R, C, G, K, M, S, or W. <220> <221> MISC_FEATURE <222> (18)..(18) <223> Amino acid X can be A, E, F, G, H, I, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (19)..(19) <223> The amino acid X can be V, A, C, D, E, F, G, I, L, P, S, T, or W. <220> <221> MISC_FEATURE <222> (20)..(20) <223> The amino acid X can be K, A, E, F, G, M, N, P, Q, R, S, T, or Y. <220> <221> MISC_FEATURE <222> (21)..(21) <223> Amino acid X can be D, A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V. W or Y <220> <221> MISC_FEATURE <222> (22)..(22) <223> The amino acid X can be H, D, E, G, M, N, P, Q, R, V, W, or Y. <220> <221> MISC_FEATURE <222> (23)..(23) <223> The amino acid X can be L, C, E, F, G, M, Q, S, V, or W. <220> <221> MISC_FEATURE <222> (24)..(24) <223> The amino acid X can be K, C, G, M, N, Q, R, S, T, or W. <220> <221> MISC_FEATURE <222> (25)..(25) <223> The amino acid X can be K, E, F, M, N, P, Q, R, S, or V. <220> <221> MISC_FEATURE <222> (26)..(26) <223> The amino acid X can be E, C, D, G, K, N, Q, R, S, V, or W. <220> <221> MISC_FEATURE <222> (27)..(27) <223> The amino acid X can be G, A, C, D, E, L, N, R, S, or V. <220> <221> MISC_FEATURE <222> (28)..(28) <223> The amino acid X can be P, A, D, E, G, K, L, M, N, Q, R, S, T, or V. <220> <221> MISC_FEATURE <222> (29)..(29) <223> The amino acid X can be H, A, C, D, E, G, I, K, L, M, N, P, Q, R, S, T, V, or Y <220> <221> MISC_FEATURE <222> (30)..(30) <223> The amino acid X can be W, C, D, G, I, L, R, S, or T. <220> <221> MISC_FEATURE <222> (31)..(31) <223> The amino acid X can be N, A, D, E, F, G, H, I, K, L, M, R, S, V, W, or Y. <220> <221> MISC_FEATURE <222> (32)..(32) <223> The amino acid X can be I, F, H, L, M, P, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (33)..(33) <223> The amino acid X can be T, A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y. <220> <221> MISC_FEATURE <222> (34)..(34) <223> The amino acid X can be S, A, D, G, K, L, M, N, P, Q, R, T, V, or W. <220> <221> MISC_FEATURE <222> (35)..(35) <223> The amino acid X can be T, A, F, G, I, K, L, N, P, Q, R, S, V, W, or Y. <220> <221> MISC_FEATURE <222> (36)..(36) <223> The amino acid X can be N, A, D, E, G, I, K, L, P, Q, R, S, T, or V. <220> <221> MISC_FEATURE <222> (37)..(37) <223> The amino acid X can be N, A, D, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (38)..(38) <223> The amino acid X can be G, A, D, E, H, I, K, L, M, P, Q, R, S, T, V, or Y. <220> <221> MISC_FEATURE <222> (39)..(39) <223> Amino acid X can be A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (40)..(40) <223> Amino acid X can be E, A, C, D, G, H, K, M, N, P, Q, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (41)..(41) <223> The amino acid X can be L, C, D, F, G, H, K, P, Q, R, S, V, or W. <220> <221> MISC_FEATURE <222> (42)..(42) <223> Amino acid X can be V, A, D, E, F, G, I, K, L, M, R, S, W, or Y. <220> <221> MISC_FEATURE <222> (43)..(43) <223> The amino acid X can be V, A, E, G, I, L, N, R, or T. <220> <221> MISC_FEATURE <222> (44)..(44) <223> The amino acid X can be R, A, E, G, I, K, L, M, Q, S, T, or W. <220> <221> MISC_FEATURE <222> (45)..(45) <223> Amino acid X can be G, A, C, D, E, K, L, M, N, Q, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (46)..(46) <223> The amino acid X can be I, A, C, E, F, G, L, M, N, S, T, or V. <220> <221> MISC_FEATURE <222> (47)..(47) <223> The amino acid X can be H, A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (48)..(48) <223> Amino acid X can be E, A, C, D, G, H, K, L, N, P, Q, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (49)..(49) <223> The amino acid X can be S, A, D, E, F, G, H, I, K, L, N, P, R, T, V, or W. <220> <221> MISC_FEATURE <222> (50)..(50) <223> Amino acid X can be D, A, E, F, G, I, K, N, Q, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (51)..(51) <223> Amino acid X can be A, E, G, R, S, T, V, or Y. <220> <221> MISC_FEATURE <222> (52)..(52) <223> The amino acid X can be K, A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (53)..(53) <223> The amino acid X can be R, A, C, D, E, G, H, I, L, M, N, Q, S, T, V, or W. <220> <221> MISC_FEATURE <222> (54)..(54) <223> The amino acid X can be I, F, M, N, or T. <220> <221> MISC_FEATURE <222> (55)..(55) <223> Amino acid X can be A, C, D, E, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (56)..(56) <223> The amino acid X can be K, A, D, E, F, G, I, L, M, N, P, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (57)..(57) <223> Amino acid X can be W, A, C, F, G, H, I, L, M, Q, R, S, V, or Y. <220> <221> MISC_FEATURE <222> (58)..(58) <223> The amino acid X can be V, A, C, D, E, G, K, L, M, Q, R, or S. <220> <221> MISC_FEATURE <222> (59)..(59) <223> Amino acid X can be E, A, C, D, G, H, I, K, L, M, N, Q, R, S, T, V, or Y. <220> <221> MISC_FEATURE <222> (60)..(60) <223> The amino acid X can be K, E, F, I, M, N, or R. <220> <221> MISC_FEATURE <222> (61)..(61) <223> The amino acid X can be K, R, A, G, Q, S, or W. <220> <221> MISC_FEATURE <222> (62)..(62) <223> The amino acid X can be F, C, G, I, L, S, V, W, or Y. <220> <221> MISC_FEATURE <222> (63)..(63) <223> The amino acid X can be P, A, C, E, F, G, H, K, L, M, N, Q, R, S, T, or W. <220> <221> MISC_FEATURE <222> (64)..(64) <223> The amino acid X can be G, A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (65)..(65) <223> The amino acid X can be V, A, D, F, G, H, I, K, L, Q, R, S, or T. <220> <221> MISC_FEATURE <222> (66)..(66) <223> Amino acid X can be H, A, C, D, F, G, K, L, N, Q, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (67)..(67) <223> The amino acid X can be T, C, I, K, L, N, R, S, V, or Y. <220> <221> MISC_FEATURE <222> (68)..(68) <223> Amino acid X can be E, A, D, F, G, K, M, N, Q, R, S, T, W, or Y. <220> <221> MISC_FEATURE <222> (69)..(69) <223> The amino acid X can be T, A, G, I, K, L, M, P, R, S, V, or W. <220> <221> MISC_FEATURE <222> (70)..(70) <223> The amino acid X can be Q, E, H, I, M, R, S, or V. <220> <221> MISC_FEATURE <222> (71)..(71) <223> The amino acid X can be Q, C, D, E, G, H, K, L, P, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (72)..(72) <223> Amino acid X can be D, A, C, E, G, L, N, Q, R, S, V, or Y. <400> 1 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa<972>65 70 <210> 2 <211> 72 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> The X amino acid can be A, C, E, K or T <220> <221> MISC_FEATURE <222> (2)..(2) <223> The X amino acid can be V, L, M or R <220> <221> MISC_FEATURE <222> (4)..(4) <223> The X amino acid can be R or S <220> <221> MISC_FEATURE <222> (5)..(5) <223> The amino acid X can be F, G, K, L, M, or R. <220> <221> MISC_FEATURE <222> (6)..(6) <223> The amino acid X can be V, A, K, or R. <220> <221> MISC_FEATURE <222> (7)..(7) <223> The amino acid X can be F, A, G, K, or R. <220> <221> MISC_FEATURE <222> (12)..(12) <223> The amino acid X can be A, G, K, or R. <220> <221> MISC_FEATURE <222> (13)..(13) <223> Amino acid X can be E, A, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (14)..(14) <223> The amino acid X can be L or S. <220> <221> MISC_FEATURE <222> (15)..(15) <223> The amino acid X can be M, K, L, R, or V. <220> <221> MISC_FEATURE <222> (16)..(16) <223> The amino acid X can be L or R. <220> <221> MISC_FEATURE <222> (18)..(18) <223> Amino acid X can be A, F, or V. <220> <221> MISC_FEATURE <222> (19)..(19) <223> Amino acid X can be V, A, C, or S. <220> <221> MISC_FEATURE <222> (20)..(20) <223> The amino acid X can be K or R. <220> <221> MISC_FEATURE <222> (21)..(21) <223> Amino acid X can be D, A, F, G, H, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (23)..(23) <223> The amino acid X can be L or V. <220> <221> MISC_FEATURE <222> (24)..(24) <223> The amino acid X can be K, G, or R. <220> <221> MISC_FEATURE <222> (26)..(26) <223> The amino acid X can be E, R, or W. <220> <221> MISC_FEATURE <222> (27)..(27) <223> The amino acid X can be G, L, or S. <220> <221> MISC_FEATURE <222> (28)..(28) <223> The amino acid X can be P, K, M, or V. <220> <221> MISC_FEATURE <222> (29)..(29) <223> The amino acid X can be H, G, L, N, R, or S. <220> <221> MISC_FEATURE <222> (31)..(31) <223> The amino acid X can be N, R, S, or W. <220> <221> MISC_FEATURE <222> (32)..(32) <223> The amino acid X can be I, F, or W. <220> <221> MISC_FEATURE <222> (33)..(33) <223> The amino acid X can be T, F, G, R, S, V, W, or Y. <220> <221> MISC_FEATURE <222> (34)..(34) <223> The amino acid X can be S, G, K, or R. <220> <221> MISC_FEATURE <222> (35)..(35) <223> The amino acid X can be T, A, G, I, P, or S. <220> <221> MISC_FEATURE <222> (36)..(36) <223> The amino acid X can be N, A, G, R, or V. <220> <221> MISC_FEATURE <222> (37)..(37) <223> The amino acid X can be N, A, G, L, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (38)..(38) <223> The amino acid X can be G, A, K, R, or S. <220> <221> MISC_FEATURE <222> (39)..(39) <223> The amino acid X can be A, G, H, K, L, N, P, R, S, T, or V. <220> <221> MISC_FEATURE <222> (40)..(40) <223> The amino acid X can be E, A, G, Q, R, S, or T. <220> <221> MISC_FEATURE <222> (42)..(42) <223> Amino acid X can be V or F. <220> <221> MISC_FEATURE <222> (44)..(44) <223> The amino acid X can be R or K. <220> <221> MISC_FEATURE <222> (45)..(45) <223> The amino acid X can be G, R, or S. <220> <221> MISC_FEATURE <222> (46)..(46) <223> Amino acid X can be I or V. <220> <221> MISC_FEATURE <222> (47)..(47) <223> The amino acid X can be H, G, P, R, or V. <220> <221> MISC_FEATURE <222> (48)..(48) <223> The amino acid X can be E, A, G, H, K, L, or R. <220> <221> MISC_FEATURE <222> (49)..(49) <223> The amino acid X can be S, D, F, I, R, or W. <220> <221> MISC_FEATURE <222> (50)..(50) <223> Amino acid X can be D, E, G, or S. <220> <221> MISC_FEATURE <222> (52)..(52) <223> The amino acid X can be K, D, F, N, Q, R, S, T, or V. <220> <221> MISC_FEATURE <222> (53)..(53) <223> The amino acid X can be R, A, D, M, Q, or V. <220> <221> MISC_FEATURE <222> (55)..(55) <223> Amino acid X can be A, E, G, S, or T. <220> <221> MISC_FEATURE <222> (56)..(56) <223> The amino acid X can be K, A, G, N, or R. <220> <221> MISC_FEATURE <222> (57)..(57) <223> The amino acid X can be W, G, or Y. <220> <221> MISC_FEATURE <222> (58)..(58) <223> The amino acid X can be V or K. <220> <221> MISC_FEATURE <222> (59)..(59) <223> The amino acid X can be E, A, G, K, L, Q, R, S, T, or V. <220> <221> MISC_FEATURE <222> (60)..(60) <223> The amino acid X can be K, I, M, or N. <220> <221> MISC_FEATURE <222> (62)..(62) <223> Amino acid X can be F or W. <220> <221> MISC_FEATURE <222> (63)..(63) <223> The amino acid X can be P, F, G, K, L, R, or S. <220> <221> MISC_FEATURE <222> (64)..(64) <223> The amino acid X can be G, Q, R, S, T, or V. <220> <221> MISC_FEATURE <222> (65)..(65) <223> The amino acid X can be either V or T. <220> <221> MISC_FEATURE <222> (66)..(66) <223> The amino acid X can be H, G, Q, or R. <220> <221> MISC_FEATURE <222> (68)..(68) <223> The amino acid X can be E or R. <220> <221> MISC_FEATURE <222> (70)..(70) <223> The amino acid X can be Q or R. <220> <221> MISC_FEATURE <222> (71)..(71) <223> The amino acid X can be Q, T, or V. <400> 2 Xaa Xaa Val Xaa Xaa Xaa Xaa Arg Gly Asp Leu Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Arg Xaa Xaa Xaa Xaa His Xaa Xaa Lys Xaa Xaa Xaa Xaa Trp Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Val Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Ala Xaa Xaa Ile Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa 50 55 60 Xaa Xaa Thr Xaa Thr Xaa Xaa Asp 65 70 <210> 3 <211> 72 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> The X amino acid can be C, A or K <220> <221> MISC_FEATURE<00^01222><222> (2)..(2) <223> The X amino acid can be V, L or R <220> <221> MISC_FEATURE <222> (5)..(5) <223> The X amino acid can be F, G, M or R <220> <221> MISC_FEATURE <222> (6)..(6) <223> The amino acid X can be V or R. <220> <221> MISC_FEATURE <222> (7)..(7) <223> The amino acid X can be F, G, or R. <220> <221> MISC_FEATURE <222> (12)..(12) <223> The amino acid X can be A, G, or K. <220> <221> MISC_FEATURE <222> (13)..(13) <223> Amino acid X can be E, A, F, G, H, I, L, P, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (14)..(14) <223> The amino acid X can be L or S. <220> <221> MISC_FEATURE <222> (15)..(15) <223> The amino acid X can be M, K, R, or V. <220> <221> MISC_FEATURE <222> (18)..(18) <223> Amino acid X can be A or V. <220> <221> MISC_FEATURE <222> (19)..(19) <223> The amino acid X can be V, A, or S. <220> <221> MISC_FEATURE <222> (21)..(21) <223> The amino acid X can be D, A, F, G, R, S, or W. <220> <221> MISC_FEATURE <222> (23)..(23) <223> The amino acid X can be L or V. <220> <221> MISC_FEATURE <222> (24)..(24) <223> The amino acid X can be K, G, or R. <220> <221> MISC_FEATURE <222> (26)..(26) <223> The amino acid X can be E or R. <220> <221> MISC_FEATURE <222> (28)..(28) <223> The amino acid X can be P, K, or V. <220> <221> MISC_FEATURE <222> (31)..(31) <223> The amino acid X can be N, R, or S. <220> <221> MISC_FEATURE <222> (32)..(32) <223> The amino acid X can be I or W. <220> <221> MISC_FEATURE <222> (33)..(33) <223> The amino acid X can be T, G, R, or V. <220> <221> MISC_FEATURE <222> (34)..(34) <223> The amino acid X can be S, G, K, or R. <220> <221> MISC_FEATURE <222> (35)..(35) <223> The amino acid X can be T, A, or G. <220> <221> MISC_FEATURE <222> (36)..(36) <223> The amino acid X can be N, G, R, or V. <220> <221> MISC_FEATURE <222> (37)..(37) <223> The amino acid X can be N, G, R, T, V, or W. <220> <221> MISC_FEATURE <222> (38)..(38) <223> The amino acid X can be G, A, K, R, or S. <220> <221> MISC_FEATURE <222> (39)..(39) <223> The amino acid X can be A, G, H, K, P, R, S, or V. <220> <221> MISC_FEATURE <222> (40)..(40) <223> The amino acid X can be E, G, R, or S. <220> <221> MISC_FEATURE <222> (45)..(45) <223> The amino acid X can be G or R. <220> <221> MISC_FEATURE <222> (47)..(47) <223> The amino acid X can be H, R, or V. <220> <221> MISC_FEATURE <222> (48)..(48) <223> The amino acid X can be E, H, or L. <220> <221> MISC_FEATURE <222> (49)..(49) <223> The amino acid X can be S, F, I, or R. <220> <221> MISC_FEATURE <222> (50)..(50) <223> Amino acid X can be D or E. <220> <221> MISC_FEATURE <222> (52)..(52) <223> The amino acid X can be K, N, or S. <220> <221> MISC_FEATURE <222> (53)..(53) <223> Amino acid X can be R, A, D, or Q. <220> <221> MISC_FEATURE <222> (55)..(55) <223> Amino acid X can be A, E, G, S, or T. <220> <221> MISC_FEATURE <222> (56)..(56) <223> The amino acid X can be K, A, G, N, or R. <220> <221> MISC_FEATURE <222> (57)..(57) <223> Amino acid X can be W or Y. <220> <221> MISC_FEATURE <222> (59)..(59) <223> The amino acid X can be E, A, G, K, L, R, T, or V. <220> <221> MISC_FEATURE <222> (60)..(60) <223> The amino acid X can be K or I. <220> <221> MISC_FEATURE <222> (62)..(62) <223> Amino acid X can be F or W. <220> <221> MISC_FEATURE <222> (63)..(63) <223> The X amino acid can be P, G, K, R or S <220> <221> MISC_FEATURE <222> (64)..(64) <223> The X amino acid can be G, Q, R, S, T or V <220> <221> MISC_FEATURE <222> (66)..(66) <223> The X amino acid can be H, G, Q or R <220> <221> MISC_FEATURE <222> (68)..(68) <223> The X amino acid can be E or R <220> <​​​​​​​​​​​​​​​​​​​​​​​​ 65 70 <210> 4 <211> 73 <212> PRT <213> Artificial synthesis <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 4 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Thr Asn Asn Gly Ala Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Arg Thr Val Glu Lys Leu Thr Asn 50 55 60 Gly Lys Ser Gln Ser Leu Val Leu Thr 65 70 <210> 5 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 5 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Thr Asn Asn Gly Ala Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Asn Trp Ala Lys Thr Tyr Ser Pro 50 55 60 Gly Gly Lys Glu Ser Tyr Thr Ile Pro 65 70 <210> 6 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 6 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Thr Asn Asn Gly Ala Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Val Glu Lys Arg Phe Pro 50 55 60 Gly Val His Thr Glu Thr Gln Gln Asp 65 70 <210> 7 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 7 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Thr Asn Asn Gly Ala Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Ala Arg Leu Lys Phe Pro 50 55 60 Gly Thr Asp Thr Arg Ile Glu Val Arg 65 70 <210> 8 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 8 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Asp Glu Ser Gly Phe Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Arg Thr Val Glu Lys Leu Thr Asn 50 55 60 Gly Lys Ser Gln Ser Leu Val Leu Thr 65 70 <210> 9 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 9 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Asp Glu Ser Gly Phe Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Asn Trp Ala Lys Thr Tyr Ser Pro 50 55 60 Gly Gly Lys Glu Ser Tyr Thr Ile Pro 65 70 <210> 10 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 10 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Asp Glu Ser Gly Phe Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Val Glu Lys Arg Phe Pro 50 55 60 Gly Val His Thr Glu Thr Gln Gln Asp 65 70 <210> 11 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 11 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Asp Glu Ser Gly Phe Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Ala Arg Leu Lys Phe Pro 50 55 60 Gly Thr Asp Thr Arg Ile Glu Val Arg 65 70 <210> 12 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 12 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Asp Thr Ser Lys Gly Ala Glu Leu Val Val Arg Gly Ile 35 40 45 His Glu Ser Asp Ala Lys Arg Ile Ala Arg Thr Val Glu Lys Leu Thr 50 55 60 Asn Gly Lys Ser Gln Ser Leu Val Leu 65 70 <210> 13 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 13 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Asp Thr Ser Lys Gly Ala Glu Leu Val Val Arg Gly Ile 35 40 45 His Glu Ser Asp Ala Lys Arg Ile Ala Asn Trp Ala Lys Thr Tyr Ser 50 55 60 Pro Gly Gly Lys Glu Ser Tyr Thr Ile 65 70 <210> 14 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 14 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Asp Thr Ser Lys Gly Ala Glu Leu Val Val Arg Gly Ile 35 40 45 His Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Val Glu Lys Arg Phe 50 55 60 Pro Gly Val His Thr Glu Thr Gln Gln 65 70 <210> 15 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 15 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Asp Thr Ser Lys Gly Ala Glu Leu Val Val Arg Gly Ile 35 40 45 His Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Ala Arg Leu Lys Phe 50 55 60 Pro Gly Thr Asp Thr Arg Ile Glu Val 65 70 <210> 16 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 16 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Val Glu Ser Ser Gln Val Glu Leu Val Val Arg Gly Ile 35 40 45 His Glu Ser Asp Ala Lys Arg Ile Ala Arg Thr Val Glu Lys Leu Thr 50 55 60 Asn Gly Lys Ser Gln Ser Leu Val Leu 65 70 <210> 17 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 17 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Val Glu Ser Ser Gln Val Glu Leu Val Val Arg Gly Ile 35 40 45 His Glu Ser Asp Ala Lys Arg Ile Ala Asn Trp Ala Lys Thr Tyr Ser 50 55 60 Pro Gly Gly Lys Glu Ser Tyr Thr Ile 65 70 <210> 18 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 18 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Val Glu Ser Ser Gln Val Glu Leu Val Val Arg Gly Ile 35 40 45 His Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Val Glu Lys Arg Phe 50 55 60 Pro Gly Val His Thr Glu Thr Gln Gln 65 70 <210> 19 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 19 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Val Glu Ser Ser Gln Val Glu Leu Val Val Arg Gly Ile 35 40 45 His Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Ala Arg Leu Lys Phe 50 55 60 Pro Gly Thr Asp Thr Arg Ile Glu Val 65 70 <210> 20 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 20 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Arg 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Thr Asn Asn Gly Ala Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Val Glu Lys Arg Phe Pro 50 55 60 Gly Val His Thr Glu Thr Gln Gln Asp 65 70 <210> twenty one <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> twenty one Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Thr Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Thr Asn Asn Gly Ala Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Val Glu Lys Arg Phe Pro 50 55 60 Gly Val His Thr Glu Thr Gln Gln Asp 65 70 <210> twenty two <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> twenty two Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Thr Asn Asn Gly Lys Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Val Glu Lys Arg Phe Pro 50 55 60 Gly Val His Thr Glu Thr Gln Gln Asp 65 70 <210> twenty three <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> twenty three Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Thr Asn Asn Gly Ala Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Val Glu Lys Arg Phe Pro 50 55 60 Arg Val His Thr Glu Thr Gln Gln Asp 65 70 <210> twenty four <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> twenty four Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Arg 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Thr Asn Asn Gly Ala Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Val Glu Lys Arg Phe Pro 50 55 60 Arg Val His Thr Glu Thr Gln Gln Asp 65 70 <210> 25 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 25 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Thr Asn Asn Gly Lys Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Val Glu Lys Arg Phe Pro 50 55 60 Arg Val His Thr Glu Thr Gln Gln Asp 65 70 <210> 26 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 26 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Thr Leu Met 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Thr Asn Asn Gly Ala Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Val Glu Lys Arg Phe Pro 50 55 60 Arg Val His Thr Glu Thr Gln Gln Asp 65 70 <210> 27 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 27 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Thr Leu Arg 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Thr Asn Asn Gly Lys Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Val Glu Lys Arg Phe Pro 50 55 60 Gly Val His Thr Glu Thr Gln Gln Asp 65 70 <210> 28 <211> 73 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 28 Met Ala Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Thr Leu Arg 1 5 10 15 Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn 20 25 30 Ile Thr Ser Thr Asn Asn Gly Lys Glu Leu Val Val Arg Gly Ile His 35 40 45 Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Val Glu Lys Arg Phe Pro 50 55 60 Arg Val His Thr Glu Thr Gln Gln Asp 65 70 <210> 29 <211> 75 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 29 Met Thr Lys Cys Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Thr 1 5 10 15 Leu Met Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His 20 25 30 Trp Asn Ile Thr Ser Thr Asn Asn Gly Ala Glu Leu Val Val Arg Gly 35 40 45 Ile His Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Val Glu Lys Arg 50 55 60 Phe Pro Gly Val His Thr Glu Thr Gln Cys Asp 65 70 75 <210> 30 <211> 75 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Optional N-terminal methionine residue <400> 30 Met Thr Lys Cys Val Val Arg Phe Val Phe Arg Gly Asp Leu Ala Glu 1 5 10 15 Leu Met Leu Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His 20 25 30 Trp Asn Ile Thr Ser Thr Asn Asn Gly Lys Glu Leu Val Val Arg Gly 35 40 45 Ile His Glu Ser Asp Ala Lys Arg Ile Ala Lys Trp Val Glu Lys Arg 50 55 60 Phe Pro Arg Val His Thr Glu Thr Gln Cys Asp 65 70 75 <210> 31 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 31 Arg Gly Asp Leu Gly Ala Leu Ala 1 5 <210> 32 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> misc_feature <222> (5)..(6) <223> X amino acid can be any naturally occurring amino acid. <400> 32 Arg Gly Asp Leu Xaa Xaa Leu 1 5 <210> 33 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> misc_feature <222> (2)..(3) <223> X amino acid can be any naturally occurring amino acid. <400> 33 Leu Xaa Xaa Leu 1 <210> 34 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 34 Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 1 5 10 15 <210> 35 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (3)..(3) <223> The amino acid X can be G or R. <220> <221> MISC_FEATURE <222> (4)..(4) <223> The amino acid X can be either V or T. <220> <221> MISC_FEATURE <222> (5)..(5) <223> Amino acid X can be H, A, C, D, F, G, K, L, N, Q, R, S, T, V, W, or Y. <400> 35 Phe Pro Xaa Xaa Xaa Thr 1 5 <210> 36 <211> 65 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 36 Ala Glu Val Arg Phe Val Phe Arg Gly Asp Leu Thr Glu Leu Met Leu 1 5 10 15 Arg Ala Val Lys Asp His Leu Lys Lys Glu Gly Pro His Trp Asn Ile[[ID=​​​​​​​​​​​​​

Claims

1. A polypeptide consisting of an amino acid sequence selected from SEQ ID NO: 25 and SEQ ID NO: 30, wherein the polypeptide is bound to α(v)β(6) integrin (avb6).

2. The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

25.

3. The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

30.

4. The polypeptide of any one of claims 1-3, wherein the polypeptide is linked to a detectable tag.

5. The polypeptide of any one of claims 1-3, wherein the polypeptide binds to avb6 with a selectivity of at least 100-fold to avb8, avb1, avb3, avb5, a5b1, a8b1 and aiibb3.

6. The polypeptide of any one of claims 1-3, wherein the avb6 is human avb6.

7. A nucleic acid encoding the polypeptide of any one of claims 1-6.

8. An expression vector, wherein the expression vector comprises the nucleic acid of claim 7 operatively linked to a control sequence.

9. A host cell, wherein the host cell comprises the nucleic acid of claim 7 and / or the expression vector of claim 8.

10. A recombinant cell, wherein the recombinant cell expresses the polypeptide of any one of claims 1-6.

11. A pharmaceutical composition comprising: (a) the polypeptide of any one of claims 1-6, the nucleic acid of claim 7, the expression vector of claim 8, the host cell of claim 9, or the recombinant cell of claim 10; and (b) A drug-acceptable carrier.

12. Use of the polypeptide of any one of claims 1-6, the nucleic acid of claim 7, the expression vector of claim 8, the host cell of claim 9, or the recombinant cell of claim 10 in the preparation of a medicament for treating pulmonary fibrosis.

13. The use according to claim 12, wherein the pulmonary fibrosis includes idiopathic pulmonary fibrosis (IPF).

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