Computational design of alpha (v) beta (6) integrin binding proteins

Abstract

To provide an alpha (v) beta (6) integrin (avb6) binding polypeptide.SOLUTION: Disclosed herein are alpha (v) beta (6) integrin (avb6) binding polypeptides and their use in the treatment and detection of tumors and in the treatment of pulmonary fibrosis. In one aspect, a polypeptide comprising a specific amino acid sequence is disclosed, wherein the polypeptide binds to alpha (v) beta (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.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application No. 62 / 925,868, filed October 25, 2019, which is incorporated herein by reference in its entirety.

[0002] Federal Funding Statement This invention was made with government support under Grant No. R01 GM092802 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention.

[0003] Sequence Listing Description A sequence listing in computer readable form is being submitted with this application by electronic submission and is incorporated by reference in its entirety. The sequence listing is contained in the file name "19-1733-PCT_Sequence-Listing_ST25.txt", created on October 21, 2020, and is 42kb in size. [Background technology]

[0004] 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. One of these integrins, avb6, is composed of av and b6 subunits that contribute to the activation of TGF-B1 / B3. avb6 expression is strictly restricted to epithelial cells. Under normal physiological conditions, avb6 expression is almost exclusively restricted to morphological changes in specific tissues during developmental stages, and, with some exceptions, is low or absent in fully differentiated epithelia. Under pathological tissue reprogramming, avb6 expression is upregulated in tumor cell migration, wound healing, and inflammation. The level of avb6 expression generally correlates with poor overall survival. Summary of the Invention

[0005] In one aspect, a polypeptide that binds to alpha(v)beta(6) integrin (avb6) is disclosed, the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3. 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, 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 Table 1, 2, or 3, and the residue in parentheses is an alternative at 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.

[0006] In another embodiment, the polypeptide comprises an amino acid sequence 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 NOs: 4 to 30. In one embodiment, residues 8 to 10 are invariant, and optionally, one, two, or three of the amino acid residues at positions 12, 13, 14, 15, 16, 17, 36, 37, 38, 39, 40, 61, 62, 63, 64, 65, and 67 are invariant. , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 are unchanged from the reference sequence, and residue numbering begins with the first amino acid after the optional N-terminal methionine residue in SEQ ID NOs: 4-28 and with the third amino acid (Cys residue) after the optional N-terminal methionine residue in SEQ ID NOs: 29-30.

[0007] In another aspect, the present disclosure provides a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-30 and 36. In one embodiment, the polypeptide is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 25, and 29-30. In another embodiment, the amino acid changes from the reference protein are conservative amino acid substitutions. In a further embodiment, the RGD sequence is unchanged. In one embodiment, residues 8-10 are unaltered, and optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 of the amino acid residues at positions 12, 13, 14, 15, 16, 17, 36, 37, 38, 39, 40, 61, 62, 63, 64, 65, and 67 are unaltered from the reference sequence, with residue numbering starting from the first amino acid after the optional N-terminal methionine residue in SEQ ID NOs: 4-28, and from the third amino acid (Cys residue) after the optional N-terminal methionine residue in SEQ ID NOs: 29-30.

[0008] In other aspects, the disclosure provides a nucleic acid encoding a polypeptide of any embodiment or combination of embodiments disclosed herein; an expression vector comprising the nucleic acid operably linked to a regulatory sequence; a host cell or recombinant comprising a nucleic acid or expression vector of any embodiment or combination of embodiments disclosed herein; and a pharmaceutical composition comprising a polypeptide, nucleic acid, expression vector, host cell, or recombinant cell of any embodiment or combination of embodiments disclosed herein and a pharmaceutically acceptable carrier.

[0009] In one aspect, the disclosure provides for the use of a polypeptide, nucleic acid, expression vector, host cell, recombinant cell, or pharmaceutical composition of 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 disclosure provides a method for treating avb6(+) tumors or pulmonary fibrosis such as idiopathic pulmonary fibrosis (IPF), comprising administering to a subject in need thereof an amount of a polypeptide, nucleic acid, expression vector, host cell, and / or pharmaceutical composition of any embodiment or combination of embodiments disclosed herein effective to treat the tumor or IPF in the subject. In a further aspect, the present disclosure provides a method for detecting an avb6(+) tumor, comprising administering to a subject suspected of having an avb6(+) tumor an amount of a polypeptide, nucleic acid, expression vector, host cell, and / or pharmaceutical composition of any embodiment or combination of embodiments disclosed herein effective to detect the tumor in the subject.

[0010] In another aspect, the present disclosure provides a method for designing an avb6-binding polypeptide, the method comprising the steps of any embodiment or combination of embodiments disclosed in this specification and the attached appendix. [Brief explanation of the drawings]

[0011] [Figures 1a-1i]a) Computational design strategy for αvβ6-binding proteins: Structure (surface view) of αvβ6 integrin complexed with TGF-β1 peptide (schematic, PDB ID 4UM9). b) Low RMSD matches to the TGF-β1 peptide were collected from the PDB database (ribbon diagram). c) Next, Rosetta™ was used to incorporate non-clashing fragments into the α / β ferredoxin fold (schematic). d) Flexible sequence design was performed in Rosetta™ while keeping the RGD-binding loop fixed. e) Next, Rosetta™ structure prediction was used to identify the sequence in which the designed structure was in the lowest energy state. f) In addition to the RGD-binding motif, two loops (loop 1 and loop 2) mediate contacts with the αv and β6 subunits. g) The canonical RGD motif in design av6_3 interacts with the receptor at the backbone level, and Asp coordinates Mg(II). h) The -LXXL (SEQ ID NO: 33) motif immediately following the RGD-binding loop fits into a hydrophobic groove in the β6 subunit. i) Additional interactions mediated by loop 1 and loop 2, which make polar contacts with the β6 and αv subunits, respectively. [Figures 2a-2k] a. Site-saturation mutagenesis analysis of the designed binders: b–e. Most of the enriched variants are charge-complementary to the receptor (see text for details). f, g. BLI titration of purified BP1 and BP2 against αvβ6. The Kd of both variants was less than 1 nM, and each titration was performed at least twice with similar results. h. Crystal structure of BP1_disulf superimposed on the designed model (binder, αvβ6). i, j. Superimposition of the designed model of the disulfide bond with the RGD loop region with crystal structure k. The A39K mutation confers specificity for αvβ6 compared to the charge-reversed αvβ8 (Glu963 for β6 and Lys902 for β8 are shown). l. Cell surface titration of BP1 and BP2 against K562 cells stably transfected with αvβ8. BP1 lacking the A39K mutation binds to αvβ8 with a Kd of approximately 7.3 nM, whereas BP2 containing the A39K mutation binds to αvβ8 with a Kd of over 500 nM. [Figure 3] TGF-β inhibition mediated by BP1 and BP2 in the TMLC assay. Both BP1 and BP2 block αvβ6-mediated TGF-β activation with similar IC50s (199 pM and 151 pM, respectively). [Figure 4a-4b] Crystal structures from the first and second design strategies: a) Crystal structure of a variant (SEQ ID NO: 36) evolved from the first design superimposed onto the design model. The first part of the crystal structure, including the RGD loop, overlays well with the design model, but the last helix of the fold makes a half turn. b) For the second generation design, the previous crystal structure was superimposed onto αβ by aligning the RGD motif. Two loops were sampled for length and conformation, resulting in a total of 16 designs ordered in two rounds. [Figure 5] Representative metal-dependent binding of the designed protein: The designed protein exhibits metal-dependent binding to αvβ6. In the absence of metal, there is no detectable binding (left panel) compared to the presence of 1 mM Ca(II) / 1 mM Mg(II) (right panel). Expression or FITC fluorescence is plotted (X axis) against binding or SAPE fluorescence (Y axis). [Figure 6] Binding of 12 clones from the second design on the yeast surface with 50 pM biotinylated αvβ6. Expression or FITC fluorescence is plotted on the X-axis, and binding or SAPE fluorescence is plotted on the Y-axis. [Figure 7] In vitro cell surface competition assay of the five strongest binders from the second round of design. av6_3 shows the highest level of selectivity for human αvβ6 compared to human αvβ8. Log concentration of binder (X-axis) is plotted against mean fluorescence intensity (Y-axis). [Figure 8] Selection scheme for the av6_3 SSM library. The first selection was performed with 200 pM αvβ6, followed by a final selection using 100 pM receptor. [Figure 9a-9b]a. SDS-PAGE gel for one-step purification of BP2_disulf from cell lysate by heat treatment. Lane 1: ladder, Lane 2: BP2_disulf crude cell lysate. Lane 3: BP2_disulf cell lysate after boiling at 85°C for 10 min. b. CD spectra of BP2_disulf before and after nebulization. DETAILED DESCRIPTION OF THE INVENTION

[0012] All references cited are incorporated herein by reference in their entirety. In this application, unless otherwise stated, techniques used may be found in any of several well-known sources. For example, 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, 2nd edition (RIFreshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, edited by E. J. Murray, The Humana Press. Inc., Clifton, NJ) and Ambion 1998 Catalog (Ambion, Austin, TX).

[0013] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0014] As used herein, "about" means + / - 5% of the stated value.

[0015] All embodiments of any aspect of this disclosure can be used in combination unless the context clearly indicates otherwise.

[0016] Unless the context clearly requires otherwise, throughout the detailed description and the claims, words like "comprise," "comprising," and the like are to be construed in the inclusive or exhaustive sense as opposed to the exclusive sense, i.e., "including but not limited to." Words using the singular or plural also include the plural and singular, respectively. Furthermore, the words "herein," "above," and "below," and words of similar import, when used in this application, refer to this application as a whole and not to any particular portions of this application.

[0017] As used herein, amino acid residues are abbreviated 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), ribonucleotides (RIs) and nucleotides (RIs) ...Asp; D), ribonucleotides (RIs) and nucleotides (RIs) are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), aspartic acid (Asp; D), aspartic acid (Asp; D), aspartic acid (Asp; D), aspartic acid (Asp; D), aspartic acid (Asp; D), aspart ), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0018] In one aspect, the present disclosure provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, or 3 (shown in Table 1, Table 2, or Table 3), where the table indicates amino acid options at each position of the polypeptide, and the polypeptide binds to alpha(v)beta(6) integrin (avb6). As disclosed in the Examples herein, the inventors designed the claimed polypeptide as an avb6 integrin-binding protein. The designed protein is hyperthermostable and binds to avb6 with high affinity. The polypeptide can be used, for example, to treat and / or detect avb6(+) tumors in vivo, block avb6-mediated TGF-B signaling in vitro, and treat pulmonary fibrosis, such as idiopathic pulmonary fibrosis (IPF). The Examples provide saturation studies to identify potential residues present at each position of the polypeptide.

[0019] Table 1 / SEQ ID NO: 1 shows amino acid residues that may be present at any position in the polypeptide, based on saturation mutagenesis studies described in the Examples below, indicated by the AA single letter code. [Table 1-1] [Table 1-2]

[0020] Table 2 / SEQ ID NO:2 shows the amino acid residues, by AA single letter code, that may be present at any position in the polypeptide, including the more concentrated mutations found in the saturation mutagenesis studies described in the Examples below. [Table 2-1] [Table 2-2]

[0021] Table 3 / SEQ ID NO:3 shows the amino acid residues, by AA single letter code, that may be present at any position in the polypeptide, including the more enriched mutations found in the saturation mutagenesis studies described in the Examples below. [Table 3-1] [Table 3-2]

[0022] 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 positions 8-10, and in this embodiment, the interface residues include an RGD motif at residues 8-10.

[0023] In various further embodiments that can be combined: 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 17 is R; the amino acid residue at position 36 is N; the amino acid residue at position 37 is N, S, or K; the amino acid residue at position 38 is G; 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 (SEQ ID NO: 35), where X is any residue listed at position 66 in Table 1, 2, or 3, and the residues in parentheses are alternatives at that position; the amino acid residue at position 65 is V, and / or The amino acid residue at position 67 is T.

[0024] In other embodiments that can be combined: 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 17 is R; the amino acid residue at position 36 is N; the amino acid residue at position 37 is N, S, or K; the amino acid residue at position 38 is G; the amino acid residue at position 39 is A, F, or K; 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 (SEQ ID NO: 35), where X is any residue listed at position 66 in Table 1, 2, or 3, and the residues in parentheses are alternatives at that position; the amino acid residue at position 65 is V, and / or The amino acid residue at position 67 is T.

[0025] As described in the Examples below, amino acid residues at positions 12 to 17, 36 to 40, 61 to 65, and 67 of the polypeptide can directly contact avb6.

[0026] In another embodiment, the polypeptide comprises an amino acid sequence 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 NOs: 4-28, where the parenthesized residues are optional. Each of these embodiments includes an optional N-terminal methionine not included in SEQ ID NOs: 1-3. Accordingly, residue numbering in SEQ ID NOs: 4-28 begins with the first amino acid after the optional N-terminal methionine residue. [Table 4-1] [Table 4-2]

[0027] AEVRFVFRGDLTELMLRAVKDHLKKEGPHWNITSRGNELEVRGSHESDAKRIQKEFPSVQSTTQA In other embodiments, the polypeptide comprises an amino acid sequence 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 NOs: 29-30, with the parenthesized residues being optional. Each of these embodiments includes an optional N-terminal methionine and two additional residues at the N-terminus that are not included in SEQ ID NOs: 1-3. Thus, the residue numbering in SEQ ID NOs: 29-30 begins with the third amino acid (Cys residue) after the optional N-terminal methionine residue. These embodiments introduce a Cys residue that allows for disulfide bonding. The introduction of disulfide bonds renders both proteins hyperthermostable, maintaining their secondary structure at 95°C under non-reducing conditions as suggested by CD spectroscopy data. [Table 5]

[0028] In one embodiment, the polypeptide is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 (E13T) and SEQ ID NO: 25 (A39KG64R).

[0029] In one embodiment, residues 8-10 (residue numbering begins with the first amino acid after the optional N-terminal methionine residue) are invariant. As noted above, interface residues for avb6 binding include positions 8-10. In another embodiment, residues 12, 13, 14, 15, 16, 17, 36, 37, 38, 39, 40, 61, 62, 63, 64, 65, and 67 (residue numbering begins with the optional residues of SEQ ID NOS: 4-28) are invariant. Of the amino acid residues in SEQ ID NOS: 29-30 (starting from the first amino acid after the optional N-terminal methionine residue and the third amino acid (Cys residue) after the optional N-terminal methionine residue), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 are unchanged from the reference sequence. As noted above, amino acid residues 12-17, 36-40, 61-65, and 67 of the polypeptide may directly contact avb6.

[0030] In another aspect, the present disclosure provides a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-30 and 36. AEVRFVFRGDLTELMLRAVKDHLKKEGPHWNITSRGNELEVRGSHESDAKRIQKEFPSVQSTTQA (SEQ ID NO: 36)

[0031] In one embodiment, the polypeptide of this aspect is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 (E13T), SEQ ID NO: 25 (A39KG64R), and 29-30.

[0032] In one embodiment, residues 8-10 of SEQ ID NOs: 4-30 (residue numbering begins with the first amino acid after the optional N-terminal methionine residue), or residues 10-12 of SEQ ID NOs: 29-30, are invariant. As noted above, interface residues for avb6 binding include positions 8-10 (or positions 10-12 of SEQ ID NOs: 29-30). In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 of the amino acid residues at positions 12, 13, 14, 15, 16, and 67 are unchanged from the reference sequence, with residue numbering starting from the first amino acid after the optional N-terminal methionine residue in SEQ ID NOS: 4-28 and the third amino acid (Cys residue) after the optional N-terminal methionine residue in SEQ ID NOS: 29-30. As noted above, amino acid residues at positions 12-17, 36-40, 61-65, and 67 of the polypeptide may directly contact avb6.

[0033] In one embodiment, in all of the above embodiments, the amino acid changes from the reference protein may be conservative amino acid substitutions.

[0034] As used herein, "conservative amino acid substitution" means: o Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Sce, Sme, Val, Ile, Leu) may only be substituted with other hydrophobic amino acids. o Hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) can only be substituted with other hydrophobic amino acids with bulky side chains. o Amino acids with positively charged side chains (Arg, His, Lys) can only be substituted with other amino acids with positively charged side chains. o Amino acids with negatively charged side chains (Asp, Glu) can only be substituted with other amino acids with negatively charged side chains. o Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) may only be substituted with other amino acids with polar, uncharged side chains.

[0035] In one embodiment, the polypeptides of the present disclosure may be linked to a detectable label. This embodiment may be useful, for example, in diagnostic uses of the polypeptides. Radiolabels, Any suitable detectable label deemed appropriate for the intended application can be used, including, but not limited to, fluorescent or luminescent proteins, avidin, biotin, or enzymes such as peroxidase.

[0036] In all embodiments, the polypeptide binds to alpha(v)beta(6) integrin (avb6), as demonstrated by biolayer interferometry using a His-tagged Ni-NTA sensor, as detailed in the Examples below. In one embodiment, the polypeptide binds to avb6 with subnanomolar binding affinity using biolayer interferometry using a His-tagged Ni-NTA sensor, as detailed in the Examples below (Table 5). In another embodiment, the polypeptide binds to avb6 with at least 100-fold selectivity over alpha(v)beta(8) integrin (avb8), alpha(v)beta(1) integrin (avb1), alpha(v)beta(3) integrin (avb3), alpha(v)beta(5) integrin (avb5), alpha5beta1 (a5b1), alpha8beta1 (a8b1), and alpha(iib)beta(3) integrin (aiibb3), using K562 cells stably transfected with the corresponding integrins.

[0037] In another aspect, the present disclosure provides nucleic acids encoding the polypeptides of any embodiment or combination of embodiments of the present disclosure. The nucleic acid sequences may include single- or double-stranded RNA or DNA, in genomic or cDNA form, or DNA-RNA hybrids, each of which may contain chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Such recombinant nucleic acid sequences may include additional sequences useful for facilitating expression and / or purification of the encoded protein, including, but not limited to, polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings herein, it will be clear to those skilled in the art which nucleic acid sequences encode the fusion proteins of the present disclosure.

[0038] In a further aspect, the present disclosure provides an expression vector comprising a nucleic acid of any aspect of the present disclosure operably linked to a suitable control sequence. An "expression vector" includes a vector in which a nucleic acid coding region or gene is operably linked to any control sequence capable of effecting expression of the gene product. A "control sequence" operably linked to a nucleic acid sequence of the present disclosure is a nucleic acid sequence capable of effecting expression of a nucleic acid molecule. Control sequences need not be contiguous with the nucleic acid sequence, so long as they function to direct its expression. Thus, for example, an intervening non-translated but transcribed sequence can be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered "operably 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, plasmid- and virus-based expression vectors. In mammalian systems, the control sequences used to drive expression of the disclosed nucleic acids can be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters, including but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organism either as an episome or by integration into the host chromosomal DNA. In various embodiments, the expression vector can comprise a plasmid, a virus-based vector, or any other suitable expression vector.

[0039] In another aspect, the present disclosure provides a method for producing a nucleic acid, an expression vector (i.e., Host cells or recombinant cells containing the expression vectors of the present disclosure (episomes or chromosomally integrated), and / or polypeptides are provided, and host cells can be either prokaryotic or eukaryotic. Cells can be transiently or stably engineered to incorporate expression vectors of the present disclosure using techniques including, but not limited to, bacterial transformation, calcium phosphate co-precipitation, electroporation, or liposome-, DEAE-dextran-, polycation-, or viral-mediated transfection.

[0040] In another aspect, the present disclosure provides: (a) a polypeptide, nucleic acid, expression vector, or host cell of any embodiment or combination of embodiments disclosed herein; (b) a pharmaceutically acceptable carrier.

[0041] Pharmaceutical compositions of the present disclosure can be used, for example, in the methods of the present disclosure described herein. In addition to a polypeptide of the present disclosure, the pharmaceutical composition can include (a) a lyoprotectant, (b) a surfactant, (c) a bulking agent, (d) a tonicity adjusting agent, (e) a stabilizer, (f) a preservative, and / or (g) a buffering agent.

[0042] In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer, or an acetate buffer. The pharmaceutical composition may also contain a lyoprotectant, such as sucrose, sorbitol, or trehalose. In certain embodiments, the pharmaceutical composition contains a preservative, such as benzalkonium chloride, benzethonium, 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 contains a bulking agent, such as glycine. In yet other embodiments, the pharmaceutical composition includes a surfactant, such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, or a combination thereof. The pharmaceutical composition may also include a tonicity adjuster, such as a compound that makes the formulation substantially isotonic or isosmotic with human blood. Exemplary tonicity adjusters include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition further comprises a stabilizer, e.g., a molecule that, when combined with the protein of interest, substantially prevents or reduces chemical and / or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0043] The polypeptide, nucleic acid, expression vector, and / or host cell may be the only active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for the intended use.

[0044] In another aspect, the present disclosure provides for the use of a polypeptide, nucleic acid, expression vector, host cell, or pharmaceutical composition of 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).

[0045] In another aspect, the disclosure provides a method for treating an avb6(+) tumor or pulmonary fibrosis, such as idiopathic pulmonary fibrosis (IPF), comprising administering to a subject in need thereof an amount of a polypeptide, nucleic acid, expression vector, host cell, and / or pharmaceutical composition of any embodiment or combination of embodiments disclosed herein effective to treat the tumor or IPF in the subject.

[0046] As detailed in the Examples, high levels of α v β6 expression is associated with poor overall survival in a wide range of cancers, including non-small cell lung cancer (NSCLC) and pancreatic cancer. v β6-mediated activation also contributes to a wide variety of fibrotic diseases and is an established target for therapeutic intervention in idiopathic pulmonary fibrosis (IPF). IPF is a rare (10–60 cases per 100,000 people), progressive fibrotic lung disease of unknown etiology, with no cure and accounting for 57% of all lung transplants. Patients suffering from acute respiratory distress syndrome (ARDS) due to the ongoing and worsening COVID-19 pandemic exhibit patchy ground-glass opacities in lung tissue and are more likely to develop pulmonary fibrosis, especially in high-risk elderly populations. SARS-CoV-2 infection, after severe lung injury, induces TGF-βm production in the lungs and lung tissue, along with other contributing factors to pulmonary fibrosis. It has recently been shown to increase RNA. Thus, in one embodiment, the subject is a human subject with IPF and infected with SARS-CoV-2.

[0047] The subject may be any suitable subject, including, but not limited to, a human subject. As used herein, "treatment" refers to achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing the onset of symptoms characteristic of the disorder; (c) suppressing the worsening of symptoms characteristic of the disorder; (d) limiting or preventing the recurrence of the disorder in patients who previously had the disorder; and / or (e) limiting or preventing the recurrence of symptoms in patients who previously showed symptoms of the disorder. Any amount of such "treatment" would be highly beneficial to a subject with avb6(+) tumor or pulmonary fibrosis.

[0048] Short serum half-life (less than 2 hours), α v The high specificity and affinity for β6 target binding, ease of manufacture using E. coli, extreme heat resistance, and aerosol formulation of the polypeptides offer significant improvements over existing therapeutics, as described in the Examples below. In contrast to antibody inhibitors, the polypeptides of the present disclosure can be formulated for tissue-specific delivery with inherently tunable serum half-lives and reduced systemic exposure (both factors expected to improve safety and reduce the potential for unwanted side effects) (e.g., aerosol α6 for IPF). v The lung retention and short serum half-life of β6 binder therapy may favor better outcomes in the definitive lung transplant setting for IPF patients).

[0049] The methods may include administration by any suitable route deemed appropriate by the attending medical professional, including but not limited to pulmonary delivery (including but not limited to inhalation and nebulization), intravenous delivery, and intramuscular delivery.

[0050] In another aspect, the present disclosure provides a method for detecting an avb6(+) tumor, the method comprising administering to a subject suspected of having an avb6(+) tumor an amount of a polypeptide, nucleic acid, expression vector, host cell, and / or pharmaceutical composition of any embodiment or combination of embodiments disclosed herein effective to detect the tumor in the subject.

[0051] In all embodiments, the subject may be any suitable subject, including, but not limited to, a mammal such as a human.

[0052] In another aspect, the present disclosure provides a method for the preparation of any embodiment or embodiments disclosed herein. Methods for designing avb6-binding polypeptides are provided, including combinatorial steps. Details are provided in the Examples below. [Example]

[0053] Integrin α v β6 is an important therapeutic target linked to TGF-β1 / β3 activation, is upregulated in a wide variety of cancers, and is a major driver of fibrotic diseases, including idiopathic pulmonary fibrosis (IPF), which may be caused by coronavirus-induced acute respiratory distress syndrome (ARDS). However, several highly specific avb6 inhibitors have been developed. They inhibit human αβ with subnanomolar affinity and >2000-fold greater specificity than other RGD (Arg-Gly-Asp)-binding integrins. v We describe the de novo design of an ultrastable inhibitory protein that binds to β6. The crystal structure of the inhibitor closely matches the design model, with affinity and specificity resulting not only from the RGD-containing loop but also from a second loop that contacts the β6 subunit. The designed inhibitor binds to the α v Inhibits β6-mediated TGF-β signaling in vitro and α vThis allows for specific targeting of β6(+) tumors in vivo. The designed inhibitors exhibited significant therapeutic efficacy against bleomycin-induced IPF in mice when administered by intraperitoneal injection and showed promising preliminary efficacy as inhaled therapeutics. Together, these results demonstrate the power of de novo protein design in creating highly specific integrin inhibitors with therapeutic potential for immuno-oncology and the treatment of pulmonary fibrosis.

[0054] Introduction α v β6 expression is upregulated during tissue reprogramming, during tumor cell migration, wound healing, and inflammation. v β6 expression is associated with poor overall survival in a wide range of cancers, including non-small cell lung cancer (NSCLC) and pancreatic cancer. v β6-mediated activation also contributes to a wide variety of fibrotic diseases and is an established target for therapeutic intervention in idiopathic pulmonary fibrosis (IPF). IPF is a rare (10–60 cases per 100,000 people), progressive fibrotic lung disease of unknown etiology, with no cure and accounting for 57% of all lung transplants. Patients suffering from acute respiratory distress syndrome (ARDS) due to the ongoing and worsening COVID-19 pandemic exhibit patchy ground-glass opacities in lung tissue and are more likely to develop pulmonary fibrosis, particularly in high-risk elderly populations. SARS-CoV-2 infection has recently been shown to increase TGF-β mRNA in the lung and lung tissue following severe lung injury, along with other contributing factors to pulmonary fibrosis.

[0055] α complexed with an RGD-containing peptide (pdb ID 4UM9) v Based on the crystal structure of β6, and similar to other structures of RGD-containing peptides bound to integrins, arginine and aspartic acid side chains form multiple hydrogen bonds to residues at the interface between the alpha and beta subunits of the integrin. The peptide C-terminal to RGD has an alpha helical turn in which two leucines fit into a hydrophobic pocket formed by a loop in the β6 subunit. We found that the α vWe sought to incorporate the RGD-containing peptide from the β6 complex structure into a de novo designed protein with desirable properties for therapeutic candidates. We began by in silico screening candidate topologies for the peptide's eight-residue extended turn conformation (RGDLGALA (SEQ ID NO: 31, Figure 1a)). We searched the PDB database for low RMSD matches to the peptide's backbone conformation and extracted segments consisting of the matched peptide and five adjacent residues at both the N- and C-termini. We then superimposed these extended fragments onto the bound peptide conformation in the complex structure and discarded fragments that clashed with the integrin at the backbone level (Figure 1b).

[0056] We found that a small α / β ferredoxin structure (Fig. 1c, d) could scaffold the binding loop without clashing with the integrin, allowing for subsequent de novo design calculations. This fold was selected for the α-protein with a ferredoxin fold. v A two-step protocol was used to design the β6 binder. In the first step, Rosetta™ was used to constrain the torsion angles of the region corresponding to the RGD peptide to those observed in the co-crystal structure, while sampling different alpha helix, beta sheet, and loop lengths, and to assemble the structure from the fragments according to the rules for building ideal proteins (Figure 1c). In the second step, the resulting ideal ferredoxin-fold structure was superimposed onto the binding loop to determine the α6 binder. v The β6 integrin was docked to form a complex, and the amino acids of the binding surface were optimized for low-energy interactions with the target. During these design calculations, the binding RGD motif was kept fixed (Figure 1d). The final design model was subjected to ab initio structure prediction tests, and the sequence in which the designed structure was in the lowest energy state was identified (Figure 1e). Unlike most previous de novo designed protein interfaces, the α vAll of the designed interactions between β6 and the designed mini-protein are mediated by loops (Figure 1f). In addition to the RGD loop, there are two other loops that contact the αv and β6 subunits. Loop 1, connecting sheet 2 and sheet 3, contacts the β6 subunit, and loop 2, connecting helix 2 and sheet 4, contacts the αv subunit (Figure 1f).

[0057] We obtained synthetic genes encoding nine designs with different helix, strand, and loop lengths (combinatorial details are provided in the Supplementary Material). Initial testing by expression of candidate binders on the yeast cell surface showed that, as expected, four designs bound fluorescently labeled α v β6 (a biotinylated variant labeled with streptavidin R-phycoerythrin conjugate (SAPE)) bound in a metal cation-dependent manner. Using the strongest binder, Design 2, as the starting template, we constructed and screened by error-prone PCR. After three rounds of yeast surface display and fluorescence-activated cell sorting (FACS), we obtained a new variant with five mutations (02_E2V_T12A_E40V_S44I_T63I). The crystal structure of this variant (SEQ ID NO: 36) was solved at 2A resolution. The portion of the crystal structure, including the RGD loop, overlaid very well with the computational design model, although a half-turn of the last helix of the fold is present. This is likely due to Phe56, which was partially exposed in the initial design model (Figure 4).

[0058] α, which has more extensive contact with integrins v To generate the β6 binder, the second design step involved superimposing the crystal structure of the first binder onto the RGD loop to create the α6 binder. v By docking the design to β6, two loop regions close to the integrin were identified. A range of lengths and conformations of the two loops were sampled, and 16 designs with loops predicted to specifically interact with integrins were selected for experimental testing. Synthetic genes encoding these 16 designs were obtained and transfected with biotinylated αβ-labeled with SAPE. vBinding was measured using yeast surface display with the β6 protein. Of the 16 ordered designs, 12 expressed well on the yeast surface, and α v We found that the binding to β6 was metal-dependent (Ca(II), Mg(II), Figure 5, FACS data). v β6 (50 pM, 100 pM, 300 pM, and 500 pM, Supplementary Information) was used to measure binding on the yeast surface. Based on yeast surface display experiments, αv6_3 binds to α v The five strongest binders (av6_3, av6_7, av6_9, av6_11, and av6_15) were expressed in E. coli and purified, and av6_3 was identified as a β6 integrin, another integrin that contributes to TGF-β activation. v Compared to β8, α v We confirmed that αv6_3 exhibited the highest level of selectivity for β6 (Figure 7). In αv6_3, the canonical RGDLXXL (SEQ ID NO: 32) motif of the TGF-β1 prodomain is incorporated into the loop connecting sheet 1 and helix 1 of the ferredoxin fold (Figure 1g). An amphipathic helix following the RGD loop is formed by the β6 subunit (Figure 1h), which mimics the binding interaction of the TGF-β1 peptide to αvβ6. Asn37 forms a hydrogen bond with Asp901 of the β6 subunit, and Arg61 of the α v It hydrogen bonds to the backbone atom connecting residues Ser756 and Ile757 of β6 (Figure 1i). All residue numbering is based on the Rosetta™ internal numbering system, starting from the first residue of the designed binder.

[0059] To investigate the sequence determinants of binding, every residue in designed av6_3 was mutated to each of the other 19 amino acids and subjected to two rounds of yeast surface display and α vFACS sorting of β6 binding was performed (FACS, Figure 8). Deep sequencing of pre- and post-selection pools identified substitutions enriched during binding selection (see Tables 1-3). v The core residues of the β6 small binder are largely conserved, suggesting that the designed residues are near-optimal for folding (Figure 2a). Given the importance of the tripeptide motif for this binding, mutations to the RGD loop are highly depleted, as expected. There are five predominantly enriched mutations at the interface: two of the mutations (E13 / A39) interact with the β6 subunit, and one mutation, M15, interacts with the α v It is located between the groove formed by the α and β6 subunits, and P63 / G64 is located between the α v The E13 residue immediately following the amphipathic helix is ​​likely to be a hydrophobic or small polar residue, possibly due to the nearby negatively charged residue on the integrin. The enriched threonine at this position is also present in the TGF-β1 peptide. Most of the other highly enriched substitutions also involve charge complementarity. M15R / K is a threonine that is highly enriched in the α v It is well within hydrogen-bonding interactions with D220 and Y250 of β6 (Fig. 2b). v Two consecutive residues (P63 and G64) on loop 2 facing the subunit are enriched as positively charged Lys or Arg residues, which are involved in the α v It is likely to form salt bridges with two acidic residues, D218 and D220, on the β6 subunit (Figure 2c and Figure 2d). The A39K substitution, facing the β6 subunit, is likely to introduce a salt bridge with Glu963 (Figure 2e). A total of nine variants of these selected substitutions were expressed, purified, and tested, either alone or in combination, using biolayer interferometry (BLI) measurements. All variants were found to be highly potent in the α v The original, unevolved av6_3 showed subnanomolar binding affinity to β6, whereas the original, unevolved av6_3 showed subnanomolar binding affinity to α with a Kd of 1.18 nM. vβ6 (see Table 5). Two high-affinity variants were selected for further characterization: BP1 (av6_3_E13T), which has a single substitution that more closely recapitulates the TGF-β1 peptide sequence, and α v BP2 (av6_3_A39KG64R) which has two substitutions that introduce positive charges that complement the negative charges of both subunits of β6 integrin. [Table 6]

[0060] Functional properties of the designed inhibitors: TGF-β is produced as an inactive complex with latency-associated peptide (LAP). v β6 binds to this inactive LAP:TGF-β complex, releasing active TGF-β, which then interacts with TGF-βRI / RII and induces downstream signaling. v β6 expression is primarily restricted to epithelial cells and, under normal physiological conditions, is largely confined to tissues undergoing morphological changes during development, with little or no expression in fully differentiated epithelia. However, in pathological conditions, α v β6 expression is upregulated during tumor cell migration, wound healing, and tissue reprogramming during inflammation, and high levels of α v β6 expression is associated with poor overall survival in a wide range of cancers, including non-small cell lung cancer (NSCLC) and pancreatic cancer. This also highlights the importance of α6 expression in immuno-oncology. v There has been considerable interest in targeting β6.

[0061] We believe that these binders are v We set out to test their ability to bind to β6(+) cells and block TGF-β-mediated downstream signaling. We generated fluorescently labeled BP1 and BP2 by conjugating Alexafluor-488 to engineered C-terminal cysteines using maleimide chemistry. The fluorescently labeled proteins were then transfected into αβ6(+) cells. vBP1 and BP2 were titrated against β6-positive human epidermoid carcinoma A431 cells. K values ​​of 167 (±0.028) pM and 30 (±0.004) pM, respectively, were obtained. d values ​​to A431 cells (data not shown).

[0062] We next examined the ability of these designed binders 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 exhibited IC values ​​of 199 pM (95% CI [119 pM, 332 pM]) and 151 pM (95% CI [79.6 pM, 284 pM]), respectively. 50 The value of α v Blocks β6-mediated TGF-β activation (Figure 3). is α v It also blocks β8-mediated TGF-β activation, whereas BP2 has no effect even at the highest concentration tested (333 ng / ml), consistent with the in vitro binding profile (FIG. 10).

[0063] BP2 binds to A431 cells with higher affinity compared to BP1, and v BP2 was chosen for further in vivo experiments because it is more specific for β6. v To investigate whether BP2 could bind to β6(+) tumors, we generated fluorescently labeled BP2 (AF680-BP2) via an engineered C-terminal cysteine ​​by chemical conjugation to Alexafluor-680 C-2 maleimide. Six to eight-week-old female athymic nude mice were transfected with A431 cells (α6(+)). v β6(+)) and HEK 293T(α v β6(-)) was injected into the left and right shoulders, respectively. When the tumors reached a diameter of 5-10 mm, the mice were injected with 1.5 nmol of AF680-BP2 protein. AF680-BP2 inhibits α v It rapidly accumulates in β6-positive tumors, reaching excellent tumor-to-muscle fluorescence contrast ratios within 3 hours after injection (data not shown). vNo detectable fluorescence was observed in β6-negative HEK-293T tumors, and α v Selectivity for β6 was demonstrated. Semi-quantitative ex vivo biodistribution analysis of AF680-BP2 was also performed. Analysis of the fluorescence intensity in different tissues revealed that α v Accumulation of AF680-BP2 in β6-positive tumors and kidneys was evident (tumor-to-kidney ratio 1:1.04, data not shown), whereas α v No significant nonspecific binding was observed, including in β6-negative tumors. These results demonstrate the efficacy of the designed binders in in vivo α v This clearly demonstrates selective targeting of β6-positive tumors. Furthermore, quantification of whole-body imaging data of AF680-BP2 after tail vein injection suggests that its serum half-life is less than approximately 2 hours (data not shown) and that its elimination may be due to glomerular filtration from the kidney into the urine and metabolic processes in the liver.

[0064] Determinants of specificity of designed binders for other RGD-binding integrins: As mentioned above, integrin α v β8 also plays a role in TGF-β1 / TGF-β3 activation. v β8 is overexpressed in T-reg cells and is essential for the suppression of T cell-mediated inflammation. For therapeutic use, α inhibition is preferred compared to global inhibition of TGF-β. v It is desirable to inhibit β6-mediated TGF-β inhibition. In BP2, loop 2 is positioned to confer specificity between the two integrins (Fig. 2j): K39 of loop 2 faces E963 of the β6 subunit and K901 of β8 (Fig. 2j). BP2 binds to the α v Identify β8 and α v β6 / α v In a cell surface binding assay using K562 cells stably expressing β8, vBP1 exhibits over 5000-fold specificity for β6. BP1, which has an alanine (A39) at this position, has much lower specificity (Figure 2k). BP1 and BP2 do not cross-react with other RGD-binding integrins, including αvβ1, αvβ3, αvβ5, α5β1, α8β1, and αiibβ3, at concentrations up to 200 nM in cell surface binding experiments using K562 cells stably transfected with different RGD-binding integrins.

[0065] To further enhance protein stability, Rosetta™ was used to scan pairs of positions in the design to introduce optimally shaped disulfide bonds, and four variants (two for each construct) were selected for experimental characterization. By size-exclusion chromatography, both types of protein, with and without disulfide bonds, elute as single monodisperse peaks. Circular dichroism (CD) spectra of the designs show two minima centered at 208 and 222 nm, consistent with a mixed alpha / beta fold (data not shown). The introduction of disulfide bonds rendered both proteins hyperthermostable, maintaining their secondary structure at 95 °C under nonreducing conditions, as suggested by the CD spectroscopy data (data not shown). Two of these proteins (hereafter referred to as BP1_disulf and BP2_disulf) were selected for further in vitro and in vivo characterization. Both BP1_disulf and BP2_disulf can be purified in a single step from E. coli cell lysates by boiling at 85°C for 10 minutes (Figure 9). BP2_disulf binds α with subnanomolar affinity. v It binds to β6, and an RGD to KGE knockout mutation abolishes binding to the receptor, confirming that the RGD loop is required for binding.

[0066] with subnanomolar affinity vThe crystal structure of BP1_disulf, a disulfide-stabilized form of BP1 that binds to β6 and does not melt at 95°C, was solved with an RMSD of XX A. The crystal structure agrees very closely with the designed model with a root-mean-square deviation (rmsd) of 0.54 Å (Fig. 2h). In the crystal structure, the disulfide bond also adopts a conformation similar to that of the designed model (Fig. 2i). Most of the core hydrophobic residues adopt a rotamer form identical to that of the designed model. Unlike most designed protein inhibitors, most of the interactions of BP1_sulf are mediated by loops. α v There are three loops that contact β6: 1) the RGD loop, 2) loop 1, and 3) loop 2 (Figure 2f). The designed RGD-binding loop is five residues long and adopts a backbone conformation that is nearly identical to that of the designed model (Figure 2j). All residues on the loop, except for arginine, adopt a rotor conformation similar to that of the designed model (Figure 2j). The LATL motif immediately after the RGD loop forms an amphipathic helix and fits into the hydrophobic groove of the β6 subunit. Previously, we have shown that the α v β6 has been shown to recognize not only the RGD loop but also the amphipathic helix formed by the LXXL (SEQ ID NO: 33) motif, which interacts exclusively with β6, providing a blueprint for ligand binding specificity and recognition beyond the RGD sequence. Loop 1, connecting sheets 2 and 3, contacts the β6 subunit and is designed as a GGGA abego type. Loop 2, connecting helix 2 and sheet 4, contacts the αv subunit, designed as a BAAB abego type (Figure 1f). In the crystal structure, both of these loops adopt a backbone conformation nearly identical to the design model (Figure 2h). In the design model of the complex, Asn37 on loop 1 hydrogen bonds with Asp901 from the β6 subunit of the receptor, forming a contact with the αv subunit. v Although it is within coordination distance to the Ca(II) atom of β6, it is vIn the designed structure, loop 1 is ideally positioned to confer specificity for the β subunit, providing easy access to inhibitors designed specifically for integrin subtypes.

[0067] BP2-disulf reduces the amount of fibrosis and restores lung function in mice challenged with bleomycin. IPF is a progressive disease characterized by the formation of scar tissue within the lungs, with a median survival of 3-5 years from diagnosis. Patients experience progressive shortness of breath and impaired pulmonary function, measured as a decline in forced vital capacity (FVC), reduced diffusion, decreased oxygenation, and ultimately respiratory failure. The progression of pulmonary fibrosis is characterized by α v This is partly due to the exacerbation of the Smad2 / 3 pathway by β6 integrin activating TGF-β. v After confirming that we could specifically block β6-mediated TGF-β signaling, we investigated the therapeutic efficacy of this molecule in bleomycin-induced pulmonary fibrosis in mice.

[0068] Male 12-week-old C57BL / 6 mice were intratracheally instilled with 50 μL of bleomycin (1 mg / kg body weight). Starting on day 7 after bleomycin instillation and ending on day 19, mice were intraperitoneally injected with a BP2_disulf binder every other day, for a total of seven treatments compared to untreated controls (data not shown). Treatment with bleomycin (BLM) and bleomycin plus BP2_disulf resulted in a weight loss (approximately 5-8% of initial body weight) compared to the NY group (data not shown). However, BP2_disulf treatment attenuated overall weight loss 14 days after lung injury compared to the BLM group (data not shown). High-resolution lung scans of the mice were obtained using a microCT scanner. The development of fibrosis was visualized in real time. Lung damage was evident as early as day 7 and became prominent on days 14 and 21 (data not shown). By day 21, BP2_disulf treatment prevented the development of large fibrotic lesions in the lung tissue, as was common in BLM mice (data not shown). Although BP2_disulf-treated mice showed damage resulting from bleomycin challenge, the lesions were not as severe as those in the BLM group. Lung morphology in BP2_disulf-treated mice showed fibrotic lesions but maintained alveolar air spaces, which were absent in bleomycin-challenged mice (data not shown). Untreated mice showed a fibrosis rate of 4.107%, bleomycin-challenged mice showed 11.01%, and BP2_disulf-treated mice showed 6.857% (data not shown). BP2_disulf and NT mice had significantly lower fibrosis rates compared to the BLM group. Tissue density frequencies were collected by dividing the Hounsefield unit intensity into bins and sampling the scans for the bin intensity frequency. The distribution of tissue density shows a shift to the right in scans of bleomycin-injured mice, indicating an increase in dense tissue compared to NT and BP2_disulf-treated mice. The distributions of BP2_disulf-treated and NT mice are comparable (data not shown).

[0069] To confirm that BP2_disulf not only reduced the amount of bleomycin-induced fibrosis but also improved overall lung function compared to the NT group, lung mechanics were measured using the FlexiVent™ FX system 21 days after bleomycin administration. Static compliance is an indicator of the lung's elastic properties and is calculated from pressure-volume loops. BP2_disulf treatment resulted in a statistically significant increase in static compliance compared to mice treated with bleomycin alone (data not shown), demonstrating similar elastic properties to untreated mice. The forced vital capacity (FVC) of BP2_disulf-treated mice was statistically significantly increased compared to BLM mice, demonstrating similar airflow to NT mice (data not shown). BP2_disulf treatment rescues the loss of lung function caused by bleomycin-induced IPF-like restrictive lung disease. Calculation of the mean PV loop showed a 100% improvement compared to bleomycin-treated mice, almost indistinguishable from NT mice (data not shown).

[0070] Consideration The designed inhibitor (BP2_disulf) described herein binds to αvβ6 with high affinity and specificity. This protein contains a single disulfide bond, is hyperthermostable, easily expressed in E. coli with high yields, and can be purified in one step from crude cell lysates by heating at 85°C (Figure 10). BP2_disulf is highly effective in a bleomycin-induced IPF mouse model (100 μg / kg). Mice treated every other day by intraperitoneal injection showed improved lung mechanics and histopathological changes. This protein has shown promising results as an inhalation therapy in a bleomycin-induced IPF model (Supplementary Information). Due to its high thermal stability, it can also be formulated as a nebulized therapeutic. BP2_disulf maintains its secondary structure after nebulization (Figure 10). This is particularly advantageous because it limits tissue-specific exposure of the binder compared to global inhibition of TGF-β, as occurs with IP injection. Its short serum half-life (less than 2 hours) and α vThe high specificity and affinity for β6 target binding, ease of manufacture using E. coli, extreme heat resistance, and amenability to aerosol formulation of the designed small binding proteins provide an improved targeted product profile as a novel candidate therapeutic for IPF. In contrast to antibody inhibitors, the α6-binding proteins disclosed herein v β6 binders can be formulated for tissue-specific delivery with inherently tunable serum half-lives and reduced systemic exposure, both factors expected to improve safety and reduce the potential for unwanted side effects (e.g., aerosolized β6 binders for IPF). v The lung retention and short serum half-life of β6-binding agents may favor better outcomes in the eventual lung transplant setting for IPF patients. The recent SARS-COV-2 pandemic also poses a serious threat to the lung health of the elderly. While the majority of affected individuals recover without major complications, patients with ARDS may develop severe lung damage. These patients are expected to develop scars / lesions and, over time, develop pulmonary fibrosis, as was the case in the previous SARS outbreak. Therefore, the de novo engineered proteins reported herein have considerable therapeutic potential not only for the treatment of IPF, but also for the progressive respiratory diseases associated with current and future coronavirus infections, as well as in cancer immunotherapy.

[0071] A frequently encountered challenge in drug discovery is targeting a single member of a large family of closely related proteins. This can be difficult to achieve with small molecules, and developing antibody panels capable of such discrimination can be challenging as a significant amount of negative selection may be required. Our structure-based de novo design strategy provides a systematic way to achieve such specificity, integrating both previously known binding motifs and entirely new interactions in an ultrastable, small scaffold, resulting in a v β6 and α v The ability to combine functionality such as the RGD loop with the specificity of an additional loop containing K39 on a minimal, stable scaffold confers higher affinity and specificity to β8. This is a major advantage of computational design over previous approaches.

[0072] material and method Computational techniques: An overview of the design protocol is provided in the text.

[0073] Yeast display: Standard yeast surface display techniques were used to screen binding and directed evolution designs. The genes encoding the designs were cloned in-frame into petcon2 with an N-terminal aga2 and a C-terminal myc tag. Surface expression of Myc was detected using an anti-C Myc antibody, and binding was confirmed using biotinylated human α v β6 was used for detection and staining with phycoerythrin-conjugated streptavidin for FACS. 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); washing buffer: 20 mM TRIS, 150 mM NaCl, pH = 8.0, 0.5% BSA, 1 mM Ca(II), and 1 mM Mg(II).

[0074] The SSM library was generated by using mutagenic primers (see below for sequences) at each position according to a previously described protocol. The resulting library was transformed into yeast using two cycles of electroporation (biological replicates). Selection was performed twice: the library was first treated with 4 μM trypsin and 0.8 μM chymotrypsin for 5 min, followed by 200 pM biotinylated α. v The second and final round of selection involved labeling with 100 pM biotinylated α6 and collecting the top 5% of binders. v β6 was used with off-rate selection. For the off-rate selection step, 500 nM of unevolved purified av6_3 was added to the cells and rotated at 37 °C for 1 hour to select for the top 1% of the binding population (Figure 8). DNA was extracted from the pre-sort and post-sort pools and barcoded. Enrichment ratios were calculated after sequencing the pools using Illumina.

[0075] Protein expression and purification: Genes encoding protein variants were sequenced as gblock gene fragments from IDT and cloned into pet29b between NdeI / XhoI restriction sites with a C-terminal His tag. All mutant protein variants were expressed in BL21(DE3*) using Studier Autoinduction technology in standard shake flasks for 36 hours at 25°C. Cells were harvested and resuspended in 20 mM Tris, 250 mM NaCl, and 20 mM imidazole (lysis buffer). Cells were lysed using a microfluidizer, and cell debris was separated by centrifugation at 24,000 g for 45 minutes. Soluble protein was first purified using a standard Ni-NTA affinity column, followed by size exclusion chromatography (S75 10 / 300 increase) on a GE Äkta pure FPLC system. The peak corresponding to the monomeric protein was collected and further verified by mass spectrometry. For the bleomycin-induced IPF model, the protein was further purified to achieve an endotoxin level of less than 5 EU / ml.

[0076] Biotinylation of designed proteins: To generate monobiotinylated proteins, an avi tag sequence (GLNDIFEAQKIEWHE; SEQ ID NO: 34) was introduced at the N-terminus of the protein. Proteins were biotinylated either by cotransformation of the protein of interest with pBirA, a vector encoding E. coli biotin ligase for in vivo biotinylation, or by using the Avity in vitro biotinylation kit with purified protein and the manufacturer's protocol. Biotinylation was further confirmed by mass spectrometry.

[0077] Structural analysis of designed proteins: To determine the crystal structure of BP1_disulf, we expressed BP1_disulf with an N-terminal TEV-cleavable his-tag. After protein expression and purification, BP1_disulf was treated with a 1 / 100 dilution of stock TEV protease and dialyzed overnight at room temperature against TBS. After complete cleavage, monitored by SDS-page gel, the protein was loaded onto 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 set up for crystallization experiments. The bound protein and BP1_disulf were crystallized by vapor diffusion at 24 °C by mixing equal volumes of reservoir solution (0.2 M KNO3, 20% PEG3350 and 0.2 M K3 citrate, 20% PEG3350, respectively). Crystals were briefly cold-immersed in reservoir solution containing 15% PEG200 and flash-frozen in liquid nitrogen. Diffraction data were collected at the GM / CA beamline of the Advanced Photon Source (APS) at -173 °C using a MAR225 CCD detector and processed using XDS. Interestingly, the diffraction data for the binding protein were initially scaled to the P6122 space group, with large Patterson peaks at the 1 / 3 and 2 / 3 c-axes, indicating two translational NCS molecules along the c-axis. A solution was found using the designed model. The model was refined with Rosetta™ and rebuilt with phenix.autobuild. Autobuild was able to reconstruct most of the sequence of the model, but the R and R were still very high at 44% / 47%, indicating a reasonably good electron density map. The data were then rescaled to the P31 space group, and a structure containing 12 molecules per asymmetric unit was refined by tetrahedral twinning. AUTOBUILD™ was used to build one-third of the sequence and was used several times during manual building in COOT and the first few iterative steps of refinement using PHENIX™ and RefMAC™. The final structure was verified using MolProbity™. [Table 7-1] [Table 7-2]

[0078] Biophysical properties of engineered proteins: Protein secondary structure and thermal stability were measured using a JASCO-1500CD instrument. For routine wavelength scans, 10–15 μM protein in TBS (20 mM TRIS, 50 mM NaCl, pH 8.0) was used. CD spectra were measured from 240–195 nm at a scan rate of 100 nm / min. For thermal melting experiments, the signal intensity at 222 nm was monitored as a function of temperature (4°C–95°C) with a temperature gradient of 2°C / min. Samples were held at the specified temperature for at least 5 s before measurement. To investigate the role of engineered disulfide bonds on stability, 1 mM TCEP was added to the protein, and thermal stability was measured under reducing conditions.

[0079] Biolayer Interferometry for Determining Protein Binding Kinetics: Data were collected on an Octet™ RED96 (Forte Bio) and processed using the instrument's software. His-tagged protein binders were immobilized on Ni-NTA Octet sensors. Different concentrations of α v The chip was immersed in a well containing β6. The association and dissociation steps were recorded at 900 and 1200 seconds, respectively. v To exclude nonspecific binding of β6, blank sensors not loaded with binding proteins were included.

[0080] Fluorescent labeling of engineered binders: For in vitro binding assays and in vivo imaging experiments, the designed binders were labeled with AlexaFluor™ 88C5 maleimide and AlexaFluor™ 80C2 maleimide (Thermo Fisher Scientific) via C-terminal single cysteine ​​variants, respectively. In a typical labeling experiment, 50–200 μM of protein was reduced with 1 mM TCEP for 30 minutes at room temperature. A 3–5 molar excess of maleimide was added to the protein solution and rotated overnight at room temperature. The reaction mixture was then purified on an S75 Increase 10 / 300 column to separate the free dye from the labeled protein. Fluorophore conjugation was further confirmed by mass spectrometry.

[0081] In vitro binding assay using fluorescently labeled binders: Epidermoid carcinoma cells (A431) and human embryonic kidney 293T cells (HEK 293T) were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37°C in a humidified atmosphere of 5% CO2. Binding assays were performed with A431 cancer cells. A431 cells were dissociated from culture flasks using enzyme-free cell dissociation buffer (Gibco). Various concentrations of AG-AF488 and E13T-AF488 were incubated with 5 x 104 A431 cells in 1x TBS containing 0.1% BSA, 1 mM Ca2+, and 1 mM Mg2+ (BTBS) for 5 h at 4°C with rotation in suspension. Sufficient incubation volume was used to avoid greater than 5% ligand depletion. After incubation, cells were washed with BTBS and analyzed by flow cytometry on an Accuri™ C6 instrument (BD Biosciences), and data were quantified using FlowJo™ software (TreeStar). Kd values ​​were determined by fitting the data to a single-site-specific binding curve using Prism™ 7 (GraphPad Software).

[0082] α by the designed inhibitors using the TMLC assay v β6-mediated TGF-β activation Inhibition: The commercial sources and descriptions of the reagents used in the TMLC assay are listed in Table 7. [Table 8]

[0083] Recombinant human TGFb1 (R&D Systems, catalog number 240-B-010) Luciferase assay system (Promega, catalog number E1501) used according to the supplier's protocol Reporter Lysis Buffer 5X (Promega E397A) 96-well cell culture plate (Costar catalog number 7107) 96-well white-bottom, white-walled, polystyrene Optiplates™ (Perkin Elmer 6005290)

[0084] antibody Anti-TGFb1, 2, 3 mIgG1 clone 1D11 (R&D Systems MAB1835-500) 0.5mg / ml Mouse IgG1 isotype control clone 11711 (R&D Systems MAB002) 0.5mg / ml Anti-av (CD51) mIgG1 clone L230 (Enzo ALX-803-304-C100) 0.1 mg / ml Anti-avb6 3G9 (in-house) hIgG1 SP16-106 10.21mg / ml NIP228 hIgG1 (3G9 isotype) (in-house) Anti-avb8 (in-house) NIP228 hIgG1 (anti-avb8 isotype) (in-house) Anti-avb6 / b8 264RAD (in-house) BPD.95 SP10-362 10.45mg / ml Detailed protocol for the TMLC assay:

[0085] Co-culture assay setup Assay medium: DMEM + 1% FBS + penicillin / streptomycin 1. Remove 1LF TMLC cells from the flask using Accutase. Wash with 10 ml of PBS and add 5 ml of Accutase / flask Incubate at 37°C for 3-5 minutes. Add assay medium and spin at 300 x g for 5 minutes. Suspend in 5 ml of assay medium and count 3.3e6 / ml.1.65e7 total Suspend in 55 ml of assay medium 2. After counting using trypan blue exclusion, cells are suspended in assay medium at a concentration of 300,000 cells / ml. 3. Add 50ul of cell suspension per well (15000 cells / well) to appropriate wells of a 96-well tissue culture plate (see plate layout). 4. Cells are left for 3 hours to allow TMLC to adhere. Prepare ab and binding protein at 5.2x final concentration. 6. Add 200 ul of ab, binding protein, or media to the appropriate wells of a 96-well deep well pp plate. 7. Prepare 2ng / ml rhTGF-b1 in assay medium. Stock is 20ug / ml: 10,000-fold dilution (1 / 100, then 1 / 100) 2ul + 198ul medium 15ul (1 / 100) + 1485ul culture medium

[0086] Prepare cells at 2x concentration. 1) Remove HeLab8 cells from the flask using Accutase. Count using trypan blue exclusion and resuspend the cells at 300,000 cells / ml. 2.52e6 / ml. 1.263e7 suspended in a total of 42.1ml 2) Harvest the appropriate number of parental K562 cells and the desired avb6 transfected cells (30 ml) and spin at 300 x g for 5 minutes. Resuspend the cell pellet in 5 ml of assay medium and count. 1.2 x 10 6 Suspend the cells at 1000µL / ml. K562 parent cells 4.25e6 / ml 2.125e7 Total 17.7ml resuspended K562avb6 2.725e6 / ml 1.36e7 Suspend in a total of 11.4ml 3) Add 200 ul of cells or media or 2x rhTGF-b1 to the appropriate wells containing binding protein, antibody, or media in the deep well pp plate (see step 6). 4) Incubate at room temperature for 15 minutes to allow binding of the binding protein / antibody to the cells / TGF-b). 5) Aspirate media and add 100 ul / well cells + / - Abs, media, or 1 ng / ml rhTGF-b1 + / - ab to appropriate wells (see plate layout). 6) Incubate the cells at 37°C, 5% CO2 for 18-20 hours before measuring luciferase activity.

[0087] Luciferase assay 1. Remove 70ul of culture supernatant using the multichannel and store in a 96-well U-bottom polypropylene plate at -80°C for later analysis of cytokine / MMP potential. 2. Wash cells twice with 200ul / well of PBS (aspirate between washes) at the risk of losing K562 cells. 3. Add 100 ul of 1x reporter lysis buffer (1 part 5x lysis buffer + 4 parts distilled water) to each well and freeze and thaw the cells in a -80°C freezer to completely lyse the cells. 4. Prepare luciferase assay buffer by thawing it to room temperature and then adding it to the lyophilized luciferase assay substrate. 5. Transfer 80 ul of cell lysate to a white-walled, clear-bottom plate and add 100 ul of luciferase assay reagent. 6. Read the signal immediately in a luminometer. Assume an ultrasensitive luminescent (96) assay

[0088] statistical analysis All values ​​were reported as mean ± SD. Data were analyzed by one-way analysis of variance followed by Tukey's post-hoc test for multiple comparisons. Analyses and graphs were performed using GraphPad™ Prism 6.0 (GraphPad, San Diego, CA, USA). Results with p-values ​​less than 0.05 were considered statistically significant.

[0089] Sequences of all designed and evolved variants reported in this paper: see Table 4 The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as those skilled in the art will recognize.

Claims

1. A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3, which binds to alpha(v)beta(6) integrin (avb6).

2. 2. The polypeptide of claim 1, wherein 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.

3. 3. The polypeptide of claim 1, wherein the amino acid residue at position 12 is A.

4. The polypeptide according to any one of claims 1 to 3, wherein the amino acid residue at position 13 is E or T.

5. The polypeptide according to any one of claims 1 to 4, wherein the amino acid residue at position 14 is L.

6. The polypeptide of any one of claims 1 to 5, wherein the amino acid residue at position 15 is M, R, or K.

7. The polypeptide according to any one of claims 1 to 6, wherein the amino acid residue at position 16 is L.

8. The polypeptide of any one of claims 1 to 7, wherein the amino acid residue at position 37 is N, S, or K.

9. The polypeptide according to any one of claims 1 to 8, wherein the amino acid residue at position 38 is G.

10. The polypeptide of any one of claims 1 to 9, wherein the amino acid residue at position 39 is A, F, or K.

11. The polypeptide according to any one of claims 1 to 10, wherein the amino acid residue at position 40 is E.

12. 12. The polypeptide of any one of claims 1 to 11, wherein the amino acid residues at positions 62 to 67 are FP(G / R)(V / T)XT (SEQ ID NO:35), where X is any residue listed at position 66 of Table 1, 2, or 3, and the residues in parentheses are alternatives at that position.

13. The polypeptide of any one of claims 1 to 12, wherein the amino acid residue at position 65 is V.

14. The polypeptide of any one of claims 1 to 13, wherein the amino acid residue at position 61 is R or K.

15. The polypeptide according to any one of claims 1 to 14, wherein the amino acid residue at position 17 is R.

16. The polypeptide according to any one of claims 1 to 15, wherein the amino acid residue at position 36 is N.

17. The polypeptide of any one of claims 1 to 16, comprising 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 NOs: 4 to 30.

18. 18. The polypeptide of claim 17, wherein residues 8-10 are invariant, and optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 of the amino acid residues at positions 12, 13, 14, 15, 16, 17, 36, 37, 38, 39, 40, 61, 62, 63, 64, 65, and 67 are invariant from the reference sequence, and wherein residue numbering begins with the first amino acid after the optional N-terminal methionine residue in SEQ ID NOs: 4-28 and with the third amino acid (Cys residue) after the optional N-terminal methionine residue in SEQ ID NOs: 29-30.

19. A polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-30 and 36.

20. 20. The polypeptide of claim 19, wherein the polypeptide is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 25, and 29-30.

21. The polypeptide of any one of claims 19 to 20, wherein the amino acid changes from the reference protein are conservative amino acid substitutions.

22. The polypeptide according to any one of claims 19 to 21, wherein the RGD sequence is invariant.

23. 22. The polypeptide of any one of claims 19-21, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 of the amino acid residues at positions 12, 13, 14, 15, 16, 17, 36, 37, 38, 39, 40, 61, 62, 63, 64, 65, and 67 are unchanged from a reference sequence selected from the group consisting of SEQ ID NOs: 4-30, and wherein residue numbering begins with the first amino acid after the optional N-terminal methionine residue in SEQ ID NOs: 4-28 and with the third amino acid (Cys residue) after the optional N-terminal methionine residue in SEQ ID NOs: 29-30.

24. The polypeptide of any one of claims 1 to 23, linked to a detectable label.

25. 25. The polypeptide of any one of claims 1 to 24, wherein the polypeptide binds to avb6, such as human avb6, with at least 100-fold selectivity over avb8, avb1, avb3, avb5, a5b1, a8b1, and aiibb3.

26. A nucleic acid encoding the polypeptide of any one of claims 1 to 25.

27. 27. An expression vector comprising the nucleic acid of claim 26 operably linked to a regulatory sequence.

28. 28. A host cell comprising a nucleic acid according to claim 26 and / or an expression vector according to claim 27.

29. A recombinant cell expressing a polypeptide according to any one of claims 1 to 25.

30. 1. A pharmaceutical composition comprising: (a) a polypeptide, nucleic acid, expression vector, host cell, or recombinant cell according to the preceding claims; (b) a pharmaceutically acceptable carrier.

31. Use of a polypeptide, nucleic acid, expression vector, host cell, recombinant cell, or pharmaceutical composition described in any one of the preceding claims for any suitable purpose, including, but not limited to, treatment and / or detection of avb6(+) tumors in vivo, blocking avb6-mediated TGF-B signaling in vitro, and treatment of pulmonary fibrosis such as idiopathic pulmonary fibrosis (IPF).

32. A method for treating an avb6(+) tumor or pulmonary fibrosis, such as idiopathic pulmonary fibrosis (IPF), comprising administering to a subject in need thereof an amount of a polypeptide, nucleic acid, expression vector, host cell, and / or pharmaceutical composition of any embodiment or combination of embodiments disclosed herein effective to treat the tumor or IPF in the subject.

33. A method for detecting an avb6(+) tumor, comprising administering to a subject suspected of having an avb6(+) tumor an amount of a polypeptide, nucleic acid, expression vector, host cell, and / or pharmaceutical composition of any embodiment or combination of embodiments disclosed herein effective to detect the tumor in the subject.

34. A method for designing an avb6 binding polypeptide, comprising the steps of any embodiment or combination of embodiments disclosed herein and in the attached appendix.

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