Long acting Anti-alphavbeta6 integrin engineered peptides

Abstract

Provided herein are anti-avb6 integrin engineered polypeptides, fusion proteins thereof with an Fc domain, and pharmaceutically acceptable compositions comprising the same. The anti-avb6 integrin engineered polypeptides of the present technology selectively bind the avb6 integrin with low-nanomolar or picomolar binding activity.

Description

WSGR Docket No. 70774-709.601LONG ACTING ANTI-ALPHA VBETA6 INTEGRIN ENGINEERED PEPTIDESCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 717,177, filed November 6, 2024, which is entirely incorporated herein by reference.BACKGROUND

[0002] 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 integrin, a member of the broader class of integrins that bind endogenous ligands with the tripeptide motif Arg-Gly-Asp (RGD-binding integrins), is responsible for activation of TGF- B1 / B3. Aberrant expression of avb6 integrin and overactivation of TGF-B1 / B3 are associated with several fibrotic diseases and cancers. Therefore, avb6 integrin serves as an attractive target for treatment of fibrotic diseases and cancer.SUMMARY

[0003] Disclosed herein in some embodiments is an anti-alphavbeta6 (avb6) integrin engineered polypeptide comprising a sequence of Formula (IA):X1X2CX4VX6FX8FRGDX13AELX17LRAX21X22X23X24X25X26X27X28X29PX31X32X33X34X35X36T NX39GX41X42LVVX46X47IX49X50X51X52AX54X5X56X57X58X59X60EX62RFPRVX68X69X70X71X72CD (IA), wherein:X1is selected from the group consisting of T, M, N, and D;X2is selected from the group consisting of D, K, L, and Q;X4is V or T;X6is R or T;X8is selected from the group consisting of V, R, and E;X13is selected from the group consisting of L, E, and T;X17is selected from the group consisting of M, Q, and E;X21is V or A;X22is Y or K;X23is E or D;X24is selected from the group consisting of E, Y, and H;X25is selected from the group consisting of I, T, and L;WSGR Docket No. 70774-709.601X26is selected from the group consisting of K, L, Q, and E;X27is selected from the group consisting of K, E, and Q;X28is E or L;X29is selected from the group consisting of G, Y, and N;X31is H or D;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is selected from the group consisting of T, E, and S;X36is selected from the group consisting of S, E, and R;X39is N or D;X41is K or R;X42is E or Q;X46is R or Q;X47is G or N;X49is selected from the group consisting of H, D, and S;X50is selected from the group consisting of E, A, and R;X51is S or E;X52is selected from the group consisting of D, K, and E;X54is selected from the group consisting of K, E, Q, and L;X55is R or E;X56is selected from the group consisting of W, L, and I;X57is A or K;X58is K or E;X59is W or R;X60is I or V;X62is K or Q;X68is selected from the group consisting of H, T, and K;X69is T or V;X70is E or T;X71is T or I; andX72is selected from the group consisting of Q, D, and E, provided that the sequence of Formula (I) comprises less than 80% identity to the sequence: TKCVVRFVFRGDLAELMLRAVKDHLKKEGPHWNITSTNNGKELVVRGIHESWSGR Docket No. 70774-709.601DAKRIAKWVEKRFPRVHTETQCD (SEQ ID NO: 1), wherein the anti-avb6 integrin engineered polypeptide is capable of binding to avb6 integrin.

[0004] Disclosed herein in some embodiments is an anti-avb6 integrin engineered polypeptide comprising a sequence of Formula (IB):X1X2CX4VTFX8FRGDX13AELX17LRAX21X22X23X24IX26EX28X29PDX32X33X34X35X36TNX39GX41X42LVVRX47IX49X50EX52AX54X55X56KEX59X60EKRFPRVX68VX70IX72CD (IB), wherein:X1is selected from the group consisting of M, N, and D;X2is D or L;X4is V or T;X8is E or R;X13is E or T;X17is E or Q;X21is V or A;X22is Y or K;X23is E or D;X24is E or Y;X26is L or Q;X28is E or L;X29is N or Y;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is E or SX36is R or E;X39is N or D;X41is K or R;X42is E or Q;X47is G or N;X49is D or S;X50is selected from the group consisting of E, A, and R;X52is selected from the group consisting of D, K, and E;X54is L or E;X55is R or E;WSGR Docket No. 70774-709.601X56is W or L;X59is W or R;X60is I or V;X68is K or T;X70is E or T; andX72is E or D, provided that the sequence of Formula (I) has less than 80% identity to the sequence: TKCVVRFVFRGDLAELMLRAVKDHLKKEGPHWNITSTNNGKELVVRGIHES DAKRIAKWVEKRFPRVHTETQCD (SEQ ID NO: 1), wherein the anti-avb6 integrin engineered polypeptide is capable of binding to avb6 integrin.

[0005] Disclosed herein in some embodiments is an anti-avb6 integrin engineered polypeptide having a half-life greater than 25 hours in plasma, the engineered polypeptide comprising a sequence of Formula (I):X1X2CX4VX6FX8FRGDX13AELX17LRAX21X22X23X24X25X26X27X28X29PX31X32X33X34X35X36T NX39GX41X42LVVX46X47IX49X50X51X52AX54X5X56X57X58X59X60EX62RFPRVX68X69X70X71X72CD (I), wherein:X1is selected from the group consisting of T, M, N, and D;X2is selected from the group consisting of D, K, L, and Q;X4is V or T;X6is R or T;X8is selected from the group consisting of V, R, and E;X13is selected from the group consisting of L, E, and T;X17is selected from the group consisting of M, Q, and E;X21is V or A;X22is Y or K;X23is E or D;X24is selected from the group consisting of E, Y, and H;X25is selected from the group consisting of I, T, and L;X26is selected from the group consisting of K, L, Q, and E;X27is selected from the group consisting of K, E, and Q;X28is E or L;X29is selected from the group consisting of G, Y, and N;X31is H or D;WSGR Docket No. 70774-709.601X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is selected from the group consisting of T, E, and S;X36is selected from the group consisting of S, E, and R;X39is N or D;X41is K or R;X42is E or Q;X46is R or Q;X47is G or N;X49is selected from the group consisting of H, D, and S;X50is selected from the group consisting of E, A, and R;X51is S or E;X52is selected from the group consisting of D, K, and E;X54is selected from the group consisting of K, E, Q, and L;X55is R or E;X56is selected from the group consisting of W, L, and I;X57is A or K;X58is K or E;X59is W or R;X60is I or V;X62is K or Q;X68is selected from the group consisting of H, T, and K;X69is T or V;X70is E or T;X71is T or I; andX72is selected from the group consisting of Q, D, and E, provided that the sequence of Formula (I) comprises less than 80% identity to the sequence: TKCVVRFVFRGDLAELMLRAVKDHLKKEGPHWNITSTNNGKELVVRGIHES DAKRIAKWVEKRFPRVHTETQCD, (SEQ ID NO: 1), wherein the anti-avb6 integrin engineered polypeptide is capable of binding to avb6 integrin.

[0006] Disclosed herein in some embodiments is an anti-avb6 integrin engineered polypeptide exhibiting reduced clearance in plasma relative to a control anti-avb6 integrinWSGR Docket No. 70774-709.601 polypeptide, the engineered polypeptide comprising an anti-avb6 integrin engineered polypeptide having a sequence of Formula (I):X1X2CX4VX6FX8FRGDX13AELX17LRAX21X22X23X24X25X26X27X28X29PX31X32X33X34X35X36TNX39GX41X42LVVX46X47IX49X50X51X52AX54X5X56X57X58X59X60EX62RFPRVX68X69X70X71X72CD (I), wherein:X1is selected from the group consisting of T, M, N, and D;X2is selected from the group consisting of D, K, L, and Q;X4is V or T;X6is R or T;X8is selected from the group consisting of V, R, and E;X13is selected from the group consisting of L, E, and T;X17is selected from the group consisting of M, Q, and E;X21is V or A;X22is Y or K;X23is E or D;X24is selected from the group consisting of E, Y, and H;X25is selected from the group consisting of I, T, and L;X26is selected from the group consisting of K, L, Q, and E;X27is selected from the group consisting of K, E, and Q;X28is E or L;X29is selected from the group consisting of G, Y, and N;X31is H or D;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is selected from the group consisting of T, E, and S;X36is selected from the group consisting of S, E, and R;X39is N or D;X41is K or R;X42is E or Q;X46is R or Q;X47is G or N;X49is selected from the group consisting of H, D, and S;WSGR Docket No. 70774-709.601X50is selected from the group consisting of E, A, and R;X51is S or E;X52is selected from the group consisting of D, K, and E;X54is selected from the group consisting of K, E, Q, and L;X55is R or E;X56is selected from the group consisting of W, L, and I;X57is A or K;X58is K or E;X59is W or R;X60is I or V;X62is K or Q;X68is selected from the group consisting of H, T, and K;X69is T or V;X70is E or T;X71is T or I; andX72is selected from the group consisting of Q, D, and E.

[0007] Disclosed herein in some embodiments is an anti-avb6 integrin engineered polypeptide exhibiting reduced clearance in plasma characterized by an AUC of at least 50,000 hr*ng / mL and a half-life in plasma greater than 25 hours, the engineered polypeptide comprising an anti-avb6 integrin engineered polypeptide having a sequence of Formula (I):X1X2CX4VX6FX8FRGDX13AELX17LRAX21X22X23X24X25X26X27X28X29PX31X32X33X34X35X36TNX39GX41X42LVVX46X47IX49X50X51X52AX54X5X56X57X58X59X60EX62RFPRVX68X69X70X71X72CD (I), wherein:X1is selected from the group consisting of T, M, N, and D;X2is selected from the group consisting of D, K, L, and Q;X4is V or T;X6is R or T;X8is selected from the group consisting of V, R, and E;X13is selected from the group consisting of L, E, and T;X17is selected from the group consisting of M, Q, and E;X21is V or A;X22is Y or K;X23is E or D;WSGR Docket No. 70774-709.601X24is selected from the group consisting of E, Y, and H;X25is selected from the group consisting of I, T, and L;X26is selected from the group consisting of K, L, Q, and E;X27is selected from the group consisting of K, E, and Q;X28is E or L;X29is selected from the group consisting of G, Y, and N;X31is H or D;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is selected from the group consisting of T, E, and S;X36is selected from the group consisting of S, E, and R;X39is N or D;X41is K or R;X42is E or Q;X46is R or Q;X47is G or N;X49is selected from the group consisting of H, D, and S;X50is selected from the group consisting of E, A, and R;X51is S or E;X52is selected from the group consisting of D, K, and E;X54is selected from the group consisting of K, E, Q, and L;X55is R or E;X56is selected from the group consisting of W, L, and I;X57is A or K;X58is K or E;X59is W or R;X60is I or V;X62is K or Q;X68is selected from the group consisting of H, T, and K;X69is T or V;X70is E or T;X71is T or I; andX72is selected from the group consisting of Q, D, and E.WSGR Docket No. 70774-709.601

[0008] In some embodiments, the anti-avb6 engineered polypeptide comprises less than 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO. 1. In some embodiments, the anti-avb6 integrin engineered polypeptide comprises at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9. In some embodiments, the anti-avb6 integrin engineered polypeptide comprises at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9. In some embodiments, the anti-avb6 integrin engineered polypeptide comprises at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9. In some embodiments, the anti-avb6 integrin engineered polypeptide comprises at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9. In some embodiments, the anti-avb6 integrin engineered polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 2-9. In some embodiments, the anti-avb6 integrin engineered polypeptide has a length of 74 amino acids. In some embodiments, the anti-avb6 integrin engineered polypeptide binds to human avb6 with a KD of less than 0.45 nM.

[0009] In some embodiments, the anti-avb6 integrin engineered polypeptide is conjugated to a signal peptide. In some embodiments, the signal peptide comprises the sequence: MARAWIFFLLCLAGRALA (SEQ ID NO: 16). In some embodiments, the signal peptide is conjugated to the N-terminus of the anti-avb6 integrin engineered polypeptide. In some embodiments, the signal peptide is conjugated to the C-terminus of the anti-avb6 integrin binding engineered polypeptide.

[0010] In some embodiments, the anti-avb6 integrin engineered polypeptide further comprises a cleavable tag. In some embodiments, the cleavable tag is located at the N-terminus of the engineered polypeptide. In some embodiments, the cleavable tag is located at the C-terminus of the engineered polypeptide. In some embodiments, the cleavable tag comprises a cleavage site and a histidine tag. In some embodiments, the histidine tag comprises the sequence HHHHHH. In some embodiments, the cleavage site comprises the sequence ENLYFQ.

[0011] In some embodiments, the signal peptide or the cleavable tag is conjugated to the anti-avb6 integrin engineered polypeptide via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a GS-rich linker. In some embodiments, the linker is GGGGS.

[0012] In some embodiments, the engineered polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 32-36.

[0013] Disclosed herein in some embodiments is a fusion protein, comprising: a) the anti-avb6 integrin engineered polypeptide as described herein, andWSGR Docket No. 70774-709.601 b) an Fc domain.

[0014] In some embodiments, the fusion protein comprises at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31. In some embodiments, the fusion protein comprises at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31. In some embodiments, the fusion protein comprises at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31. In some embodiments, the fusion protein comprises at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31. In some embodiments, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 17-31. In some embodiments, the fusion protein comprises a sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 17- 22, and 24-36. In some embodiments, the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 17-22, and 24-36. In some embodiments, the fusion protein comprises a sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 21.

[0015] In some embodiments, the fusion protein exhibits reduced clearance in plasma characterized by an AUC of at least 50,000 hr*ng / mL and a half-life greater than 25 hours in plasma. In some embodiments, the fusion protein exhibits reduced clearance in plasma relative to a control anti-avb6 integrin polypeptide not conjugated to the Fc domain.In some embodiments, the fusion protein comprises an Fc domain. In some embodiments, the Fc domain is a modified Fc domain comprising one or more amino acid substitutions relative to a wild type Fc domain. In some embodiments, the modified Fc domain comprises amino acid substitutions M252Y, S254T, and T256E (YTE mutation), according to EU numbering, compared with a wild type Fc domain. In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the Fc domain comprises a sequence comprising at 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-15: In some embodiments, the Fc domain comprises a sequence selected from the group consisting of SEQ ID NOs: 10-15:

[0016] In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises the sequence: MARAWIFFLLCLAGRALA (SEQ ID NO: 16).

[0017] In some embodiments, the signal peptide is conjugated to the N-terminus of the Fc domain.WSGR Docket No. 70774-709.601

[0018] In some embodiments, the signal peptide is conjugated to the C-terminus of the Fc domain.

[0019] In some embodiments, the signal peptide is conjugated to the N-terminus of the anti- avb6 integrin binding engineered polypeptide. In some embodiments, the signal peptide is conjugated to the C-terminus of the anti-avb6 integrin binding engineered polypeptide. In some embodiments, the anti-avb6 integrin binding engineered polypeptide is conjugated to the C- terminus of the Fc domain.

[0020] In some embodiments, the anti-avb6 integrin binding engineered polypeptide is conjugated to the Fc domain via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a GS-rich linker. In some embodiments, the linker is GGGGS or GGSG.

[0021] In some embodiments, the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 17-26.

[0022] In some embodiments, the anti-avb6 integrin engineered polypeptide is conjugated to the N-terminus of the Fc domain. In some embodiments, the anti-avb6 integrin engineered polypeptide is conjugated to the N-terminus of the Fc domain via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a G- or GS-rich linker. In some embodiments, the linker comprises a sequence selected from the group consisting of: G, GGGGS, GGGGSGGGGS, and GGGGS GGGGS GGGG. In some embodiments, the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 27-30.

[0023] In some embodiments, the fusion protein comprises two or more anti-avb6 integrin binding engineered polypeptides conjugated to the Fc domain. In some embodiments, one of the two or more anti-avb6 integrin binding engineered polypeptides is conjugated to the C-terminus of the Fc domain and the other of the two or more anti-avb6 integrin binding engineered polypeptides is conjugated to the N-terminus of the Fc domain. In some embodiments, the two or more anti-avb6 integrin binding engineered polypeptides comprise the same sequence. In some embodiments, the two or more anti-avb6 integrin binding engineered polypeptides are conjugated to the Fc domain via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises a GS-rich linker. In some embodiments, the peptide linker is GGGGS. In some embodiments, the fusion protein comprises the sequence of SEQ ID NO: 31. In some embodiments, the fusion protein is a homodimer of the Fc domain and the anti- avb6 integrin engineered polypeptide.

[0024] In some embodiments, the fusion protein exhibits reduced clearance in plasma relative to a control anti-avb6 integrin engineered polypeptide or a control protein. In someWSGR Docket No. 70774-709.601 embodiments, the fusion protein exhibits increased AUC in plasma relative to a control anti-avb6 integrin engineered polypeptide or a control protein. In some embodiments, the fusion protein exhibits increased half-life in plasma relative to a control anti-avb6 integrin engineered polypeptide or a control protein. In some embodiments, the fusion protein exhibits one or more of the following, relative to a control anti-avb6 integrin engineered polypeptide or a control fusion protein: (a) an increase in half-life by at least about 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1,000%, 2,000%, 3,000% 4,000%, 5,000%, 6,000%, 7,000%, 8,000%, 9,000%, 10,000%, 11,000%, 12,000%, 13,000%, 14,000%, or 15,000%; (b) a reduction in clearance by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%; and (c) an increase in an AUC by at least about 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1,000%, 2,000%, 3,000% 4,000%, 5,000%, 6,000%, 7,000%, 8,000%, 9,000%, 10,000%, 11,000%, 12,000%, 13,000%, 14,000%, or 15,000%.

[0025] In some embodiments, the control protein is motavizumab-YTE.

[0026] In some embodiments, the fusion protein exhibits reduced clearance in plasma characterized by an AUC of at least 50,000 h*ng / mL. In some embodiments, the fusion protein exhibits reduced clearance in plasma characterized by an AUC of at least 1,000,000 h*ng / mL. In some embodiments, the fusion protein exhibits reduced clearance in plasma characterized by a half-life of at least 25 hours. In some embodiments, the fusion protein exhibits reduced clearance in plasma characterized by a half-life of at least about 30 hours. In some embodiments, the sequence identity is determined by a BLASTP, CLUSTALW, MUSCLE, MAFFT algorithm, or a CLUSTALW algorithm with the Smith-Waterman homology search algorithm parameters.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 illustrates plasma concentrations of anti-avb6 integrin engineered polypeptides and fusion proteins of the present technology following intravenous administration of SEQ ID NOs: 17-22 and a control (Motavizumab-YTE) to wild-type (WT) mice at 5 mg / kg.

[0028] FIGS. 2A-2C illustrates plasma concentrations of fusion proteins of the present technology following intravenous administration of SEQ ID NOs: 18, 21, 22, 23, and 26, and a control (Motavizumab-YTE), to WT mice at 5 mg / kg.

[0029] FIG. 3 illustrates plasma concentrations of fusion proteins of the present technology following intravenous or subcutaneous administration of SEQ ID NOs: 21 and 23 to WT mice at 5 mg / kg.WSGR Docket No. 70774-709.601

[0030] FIGS. 4A-4C illustrate plasma concentrations, AUC, and T1 / 2 of fusion proteins comprising different linkers of the present technology following intravenous administration of SEQ ID NOs: 18, 22 and 27-31, and a control (Motavizumab-YTE), to WT mice at 5 mg / kg.

[0031] FIGS. 5A and 5B illustrate plasma concentrations of fusion proteins of the present technology following intravenous administration of SEQ ID NO: 21, and a control (Motavizumab- YTE), to Tg32 mice at 5 mg / kg.

[0032] FIG. 6 illustrates plasma concentrations of fusion proteins of the present technology following intravenous administration of SEQ ID NO: 21 to Cynomolgus Monkeys at 5 mg / kg.DETAILED DESCRIPTION

[0033] The present technology provides anti-avb6 integrin engineered polypeptides having extended activity in vivo that selectively bind avb6 integrin. The extended activity of the anti- avb6 integrin engineered polypeptides may be achieved through reduced clearance of the engineered polypeptides, as characterized by extended half-life and increased AUC. As a consequence of the extended activity, the anti-avb6 integrin engineered polypeptides may be administered to a subject less frequently than a control avb6 integrin binding polypeptide resulting in a long-acting anti-avb6 integrin engineered polypeptide. For example, as aberrant avb6 expression is associated with a wide range of fibrotic diseases, the anti-avb6 integrin engineered polypeptides of the present technology may be administered to treat and / or prevent one or more fibrotic diseases at a less frequent dose than a control avb6 integrin binding polypeptide administered for the treatment and / or prevention of the one or more fibrotic diseases.

[0034] Anti-avb6 integrin engineered polypeptides of the present technology include an anti-avb6 integrin engineered polypeptide that selectively binds avb6 over other RGD-binding integrins, such as, for example, avbl, avb3, avb5, a5bl, and avb8 integrins. For example, the anti- avb6 integrin engineered polypeptide may bind avb6 integrin with low nanomolar to picomolar affinity. The anti-avb6 integrin engineered polypeptide may be conjugated to a functional domain that enables the extended activity of the anti-avb6 integrin engineered polypeptides.

[0035] The following description is merely exemplary in nature and is not intended to limit the present technology, its applications, or its uses. It should be understood that throughout the drawings, corresponding reference numerals indicate like or corresponding parts and features. The description of specific examples indicated in various embodiments of the present technology are intended for purposes of illustration only and are not intended to limit the scope of the present technology disclosed herein. Moreover, recitation of multiple embodiments having stated featuresWSGR Docket No. 70774-709.601 is not intended to exclude other embodiments having additional features or other embodiments incorporating different combinations of the stated features.

[0036] Furthermore, the detailed description of various embodiments herein makes reference to the accompanying drawing / FIGS, which show various embodiments by way of illustration. While the embodiments are described in sufficient detail to enable those skilled in the art to practice the present technology, it should be understood that other embodiments may be realized, and that structural changes may be made without departing from the spirit and scope of the present technology. Thus, the detailed description herein is presented for purposes of illustration only and not of limitation.Definitions

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present technology belongs. For the purposes of the present technology, the following terms are defined below.

[0038] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0039] The term “about” means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by acceptable levels in the art. Typically, such variation may be as much 10% above and below a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length and such variation may be influenced by standard applicable measurement practices. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth.

[0040] As used herein, a “composition” or a “pharmaceutical composition” refers to a mixture of the active ingredient with other chemical components, such as pharmaceutically acceptable carriers and / or excipients.

[0041] As used herein, a “pharmaceutically acceptable carrier” of the pharmaceutical composition refers to a carrier or diluent that does not cause significant irritation to an organism, does not abrogate the biological activity and properties of the administered active ingredient, and / or does not interact in a deleterious manner with the other components of the composition in which it is contained. The term “carrier” encompasses any excipient, binder, diluent, filler, salt,WSGR Docket No. 70774-709.601 buffer, solubilizer, lipid, stabilizer, or other material for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005, which is incorporated herein by reference in its entirety). Some examples of physiologically acceptable carriers include antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN® (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, N.J.). An “excipient” of the pharmaceutical composition refers to an inert substance added to a composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0042] An “anti-avb6 integrin engineered polypeptide” and “engineered polypeptides” are used interchangeably to refer to an engineered polypeptide that opposes the avb6 integrin- associated responses normally induced by an avb6 integrin agonist agent.

[0043] A “control avb6 integrin binding polypeptide” or control polypeptide as used herein refers to a polypeptide that binds to avb6 integrin with low nanomolar or picomolar binding affinity, such as, for example, a polypeptide that binds avb6 integrin with a KD of less than 1 nM. In some embodiments, the control avb6 integrin binding polypeptide is SEQ ID NO: 1.

[0044] The terms “bind,” “binding,” “complex,” and “complexing,” refer to all types of physical and chemical binding, reactions, complexing, attraction, chelating and the like.

[0045] The peptides or polypeptides described herein can be (a) naturally occurring, (b) produced by chemical synthesis, (c) produced by recombinant DNA technology, (d) produced by biochemical or enzymatic fragmentation of larger molecules, (e) produced by methods resulting from a combination of methods (a) through (d) listed above, or (f) produced by any other means for producing polypeptides.WSGR Docket No. 70774-709.601

[0046] As used herein, the terms “host cell” and “recipient cell” are intended to include any individual cell or cell culture that can be or has / have been recipients of vectors, exogenous nucleic acid molecules, and polynucleotides encoding the polypeptide or binding agent of the present disclosure; and / or recipients of the polypeptide or binding agent itself. The introduction of the respective material into the cell is carried out by way of transformation, transfection and the like. The term “host cell” is also intended to include progeny or potential progeny of a single cell. Because certain modifications may occur in succeeding generations due to either natural, accidental, or deliberate mutation or due to environmental influences, such progeny may not, in fact, be completely identical (in morphology or in genomic or total DNA complement) to the parent cell but is still included within the scope of the term as used herein. Suitable host cells include prokaryotic or eukaryotic cells, and also include but are not limited to bacteria, yeast cells, fungi cells, plant cells, and animal cells such as insect cells and mammalian cells, e.g., murine, rat, macaque or human.

[0047] A “vector” is a nucleic acid molecule used as a vehicle to transfer (foreign) genetic material into a cell. The term “vector” encompasses — but is not restricted to — plasmids, viruses, cosmids and artificial chromosomes. In general, engineered vectors comprise an origin of replication, a multicloning site and a selectable marker. The vector itself is generally a nucleotide sequence, commonly a DNA sequence, that comprises an insert (transgene) and a larger sequence that serves as the “backbone” of the vector. Modem vectors may encompass additional features besides the transgene insert and a backbone: promoter, genetic marker, antibiotic resistance, reporter gene, targeting sequence, protein purification tag. Vectors called expression vectors (expression constructs) specifically are for the expression of the transgene in the target cell, and generally have control sequences.

[0048] As used herein, the term “culturing” refers to the in vitro maintenance, differentiation, growth, proliferation and / or propagation of cells under suitable conditions in a medium.

[0049] The term “expression” includes any step involved in the production of a polypeptide or protein of the disclosure including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0050] Transfection” is the process of deliberately introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. Transfection of animal cells typically involves opening transient pores or “holes” in the cell membrane, to allow the uptake of material. Transfection can be carried out using calcium phosphate, by electroporation, by cell squeezing orWSGR Docket No. 70774-709.601 by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell membrane and deposit their cargo inside.

[0051] The term “peptide” or “polypeptide” as used herein includes any structure comprised of two or more amino acids, including chemical modifications and derivatives of amino acids. The amino acids forming all or a part of a peptide or polypeptide may be naturally occurring amino acids, stereoisomers and modifications of such amino acids, non-protein amino acids, post- translationally modified amino acids, enzymatically modified amino acids, constructs or structures designed to mimic amino acids, and the like, so that the term peptide or polypeptide includes pseudopeptides and peptidomimetics, including structures which have a non-peptidic backbone. Peptide or polypeptide also includes dimers or multimers of peptides. A “manufactured” peptide or polypeptide includes a peptide or polypeptide produced by chemical synthesis, recombinant DNA technology, biochemical, or enzymatic fragmentation of larger molecules, combinations of the foregoing or, in general, made by any other method. Peptides or polypeptides of the disclosure can contain a variable number of amino acid residues, optionally with non-amino acid residue groups at the N- and C-termini, such groups including acyl, acetyl, alkenyl, alkyl, N-alkyl, amine, or amide groups, among others.

[0052] By employing chemical synthesis, a useful means of production, it is possible to introduce various amino acids which do not naturally occur along the chain, modify the N- or C- terminus, and the like, thereby providing for improved stability and formulation, resistance to protease degradation, and the like.

[0053] “Amino acids” are molecules containing an amine group, a carboxylic acid group, and a side-chain that is specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen and have the generic formula H2N — CHR — COOH, wherein R represents a side chain group. The various a-amino acids differ in the side-chain moiety that is attached to the a-carbon. The “amino acids” of the present technology include the known naturally occurring protein amino acids, which are referred to by both their common three letter abbreviation and single letter abbreviation. See generally Synthetic Peptides: A User ’s Guide, G. A. Grant, editor, W.H. Freeman & Co., New York (1992), the teachings of which are incorporated herein by reference, including the text and table set forth at pages 11 through 24. As set forth above, the term “amino acid” also includes stereoisomers and modifications of naturally occurring protein amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids, and the like. Modified and unusual amino acids are described generallyWSGR Docket No. 70774-709.601 in Synthetic Peptides: A User ’s Guide, supra; Hruby et al., Biochem. J. 268:249-262 (1990); and Toniolo, Int. J. Peptide Protein Res. 35:287-300 (1990); the teachings of all of which are incorporated herein by reference.

[0054] In the peptides or polypeptides described herein, conventional amino acid residues have their conventional meaning as given in Chapter 2400, of the Manual of Patent Examining Procedure, 8th Ed. Thus, “A” is alanine; “R” is arginine; “N” is asparagine; “D” is aspartic acid; “C” is cysteine; “Q” is glutamine; “E” is glutamic acid; “G” is glycine; “H” is histidine; “I” is isoleucine; “L” is leucine; “K” is lysine; “M” is methionine; “F” is phenylalanine; “P” is proline; “S” is serine; “T” is threonine; “W” is tryptophan; “Y” is tryosine; and “V” is valine. Unless otherwise indicated, all amino acids abbreviations represent either isomer, i.e., the L-isomer, the D-isomer, or combinations thereof can be used. Thus, for example, “L-F” or “IF” is L- phenylalanine; “D-F” or “dF” is D-phenylalanine; “D- / L-F” or “d / lF” is D-phenylalanine, L- phenylalanine, or combinations thereof; “F” is also D-phenylalanine, L-phenylalanine, or combinations thereof, and so on.

[0055] Amino acids, including stereoisomers and modifications of naturally occurring amino acids, protein amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs, or structures designed to mimic amino acids (peptide mimetics), and the like, including all of the foregoing, are sometimes referred to herein as “residues.”

[0056] The disclosure of all publications, patents, and published patent applications listed herein are hereby incorporated by reference in their entireties.Anti-avb6 Integrin Engineered Polypeptides

[0057] The present technology provides anti-avb6 integrin engineered polypeptides including an anti-avb6 integrin engineered polypeptide. The anti-avb6 integrin engineered polypeptide may be selective for avb6 over other RGD-binding integrins, such as, for example, avbl, avb2, avb5, and avb8 integrins. In some embodiments, the anti-avb6 integrin engineered polypeptide inhibits the activity of avb6 integrin.

[0058] In some embodiments, the anti-avb6 integrin engineered polypeptides comprise an anti-avb6 integrin engineered polypeptide has length of at least 70 amino acids. For example, the anti-avb6 integrin engineered polypeptide may have a length of about 70 amino acids to about 80 amino acids in length. In some embodiments, the anti-avb6 integrin engineered polypeptide hasWSGR Docket No. 70774-709.601 a length of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acids. In further embodiments, the anti-avb6 integrin engineered polypeptide has a length of 74 amino acids.

[0059] In some embodiments, the anti-avb6 integrin engineered polypeptides comprise an anti-avb6 integrin engineered polypeptide having a sequence according to Formula (I):X1X2CX4VX6FX8FRGDX13AELX17LRAX21X22X23X24X25X26X27X28X29PX31X32X33X34X35X36TNX39GX41X42LVVX46X47IX49X50X51X52AX54X55X56X57X58X59X60EX62RFPRVX68X69X70X71X72CD (I), wherein:X1is selected from the group consisting of T, M, N, and D;X2is selected from the group consisting of D, K, L, and Q;X4is V or T;X6is R or T;X8is selected from the group consisting of V, R, and E;X13is selected from the group consisting of L, E, and T;X17is selected from the group consisting of M, Q, and E;X21is V or A;X22is Y or K;X23is E or D;X24is selected from the group consisting of E, Y, and H;X25is selected from the group consisting of I, T, and L;X26is selected from the group consisting of K, L, Q, and E;X27is selected from the group consisting of K, E, and Q;X28is E or L;X29is selected from the group consisting of G, Y, and N;X31is H or D;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is selected from the group consisting of T, E, and S;X36is selected from the group consisting of S, E, and R;X39is N or D;X41is K or R;X42is E or Q;X46is R or Q;WSGR Docket No. 70774-709.601X47is G or N;X49is selected from the group consisting of H, D, and S;X50is selected from the group consisting of E, A, and R;X51is S or E;X52is selected from the group consisting of D, K, and E;X54is selected from the group consisting of K, E, Q, and L;X55is R or E;X56is selected from the group consisting of W, L, and I;X57is A or K;X58is K or E;X59is W or R;X60is I or V;X62is K or Q;X68is selected from the group consisting of H, T, and K;X69is T or V;X70is E or T;X71is T or I; andX72is selected from the group consisting of Q, D, and E.

[0060] In some embodiments, the anti-avb6 integrin engineered polypeptide has a sequence having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence: TKCVVRFVFRGDLAELMLRAVI<DHLI<I<EGPHWNITSTNNGI<ELVVRGIHESDAI<RIAI<WVEI<RFPRVHTET QCD (SEQ ID NO: 1). In some embodiments, the anti-avb6 integrin engineered polypeptide has a sequence having less than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, identity to the sequence: TKCVVRFVFRGDLAE LMLRAVKDHLKKEGPHWNITSTNNGKELVVRGIHESDAKRIAKWVEKRFPRVHTETQC D (SEQ ID NO: 1). In some embodiments, the sequence of Formula (I) has less than 80% identity to the sequence:TKCVVRFVFRGDLAELMLRAVKDHLKKEGPHWNITSTNNGKELVVRGIHES DAKRIAKWVEKRFPRVHTETQCD, wherein the anti-avb6 integrin engineered polypeptide is capable of binding to avb6 integrin.

[0061] Accordingly, in some embodiments, the anti-avb6 integrin engineered polypeptide comprises an anti-avb6 integrin engineered polypeptide having a sequence of Formula (IA):WSGR Docket No. 70774-709.601X1X2CX4VX6FX8FRGDX13AELX17LRAX21X22X23X24X25X26X27X28X29PX31X32X33X34X35X36TNX39GX41X42LVVX46X47IX49X50X51X52AX54X5X56X57X58X59X60EX62RFPRVX68X69X70X71X72CD (IA), wherein:X1is selected from the group consisting of T, M, N, and D;X2is selected from the group consisting of D, K, L, and Q;X4is V or T;X6is R or T;X8is selected from the group consisting of V, R, and E;X13is selected from the group consisting of L, E, and T;X17is selected from the group consisting of M, Q, and E;X21is V or A;X22is Y or K;X23is E or D;X24is selected from the group consisting of E, Y, and H;X25is selected from the group consisting of I, T, and L;X26is selected from the group consisting of K, L, Q, and E;X27is selected from the group consisting of K, E, and Q;X28is E or L;X29is selected from the group consisting of G, Y, and N;X31is H or D;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is selected from the group consisting of T, E, and S;X36is selected from the group consisting of S, E, and R;X39is N or D;X41is K or R;X42is E or Q;X46is R or Q;X47is G or N;X49is selected from the group consisting of H, D, and S;X50is selected from the group consisting of E, A, and R;X51is S or E;WSGR Docket No. 70774-709.601X52is selected from the group consisting of D, K, and E;X54is selected from the group consisting of K, E, Q, and L;X55is R or E;X56is selected from the group consisting of W, L, and I;X57is A or K;X58is K or E;X59is W or R;X60is I or V;X62is K or Q;X68is selected from the group consisting of H, T, and K;X69is T or V;X70is E or T;X71is T or I; andX72is selected from the group consisting of Q, D, and E, provided that the sequence of Formula (IA) has less than 80% identity to the sequence: TKCVVRFVFRGDLAELMLRAVKDHLKKEGPHWNITSTNNGKELVVRGIHESD AI<RIAI<WVEI<RFPRVHTETQCD (SEQ ID NO: 1).

[0062] In some embodiments, when the anti-avb6 integrin engineered polypeptide has a sequence of Formula (I) or (IA), X6is T, X25is I, X27is E, X31is D, X46is R, X51is E, X57is K, X58is E, X62is K, X69is V, and X71is I. In further embodiments, X1is selected from the group consisting of M, N, and D; X2is D or L; X8is E or R; X13is E or T; X17is E or Q; X24is E or Y; X26is L or Q; X29is N or Y; X35is E or S; X36is R or E; X49is D or S; X54is L or E; X56is W or L; X68is K or T; and X72is D or E.

[0063] In some embodiments, the anti-avb6 integrin engineered polypeptide comprises an anti-avb6 integrin engineered polypeptide having a sequence of Formula (IB):X1X2CX4VTFX8FRGDX13AELX17LRAX21X22X23X24IX26EX28X29PDX32X33X34X35X36TNX39GX41X42LVVRX47IX49X50EX52AX54X55X56KEX59X60EKRFPRVX68VX70IX72CD (IB), wherein:X1is selected from the group consisting of M, N, and D;X2is D or L;X4is V or T;X8is E or R;X13is E or T;WSGR Docket No. 70774-709.601X17is E or Q;X21is V or A;X22is Y or K;X23is E or D;X24is E or Y;X26is L or Q;X28is E or L;X29is N or Y;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is E or SX36is R or E;X39is N or D;X41is K or R;X42is E or Q;X47is G or N;X49is D or S;X50is selected from the group consisting of E, A, and R;X52is selected from the group consisting of D, K, and E;X54is L or E;X55is R or E;X56is W or L;X59is W or R;X60is I or V;X68is K or T;X70is E or T; andX72is E or D, provided that the sequence of Formula (I) has less than 80% identity to the sequence: TKCVVRFVFRGDLAELMLRAVKDHLKKEGPHWNITSTNNGKELVVRGIHES DAKRIAKWVEKRFPRVHTETQCD, wherein the anti-avb6 integrin engineered polypeptide is capable of binding to avb6 integrin.

[0064] In some embodiments, the anti-avb6 integrin engineered polypeptide exhibits reduced clearance in plasma characterized by an AUC of at least 50,000 hr*ng / mL and a half-lifeWSGR Docket No. 70774-709.601 in plasma greater than 25 hours, wherein the engineered polypeptide comprises an anti-avb6 integrin engineered polypeptide having a sequence of Formula (I):X1X2CX4VX6FX8FRGDX13AELX17LRAX21X22X23X24X25X26X27X28X29PX31X32X33X34X35X36TNX39GX41X42LVVX46X47IX49X50X51X52AX54X5X56X57X58X59X60EX62RFPRVX68X69X70X71X72CD (I), wherein:X1is selected from the group consisting of T, M, N, and D;X2is selected from the group consisting of D, K, L, and Q;X4is V or T;X6is R or T;X8is selected from the group consisting of V, R, and E;X13is selected from the group consisting of L, E, and T;X17is selected from the group consisting of M, Q, and E;X21is V or A;X22is Y or K;X23is E or D;X24is selected from the group consisting of E, Y, and H;X25is selected from the group consisting of I, T, and L;X26is selected from the group consisting of K, L, Q, and E;X27is selected from the group consisting of K, E, and Q;X28is E or L;X29is selected from the group consisting of G, Y, and N;X31is H or D;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is selected from the group consisting of T, E, and S;X36is selected from the group consisting of S, E, and R;X39is N or D;X41is K or R;X42is E or Q;X46is R or Q;X47is G or N;X49is selected from the group consisting of H, D, and S;WSGR Docket No. 70774-709.601X50is selected from the group consisting of E, A, and R;X51is S or E;X52is selected from the group consisting of D, K, and E;X54is selected from the group consisting of K, E, Q, and L;X55is R or E;X56is selected from the group consisting of W, L, and I;X57is A or K;X58is K or E;X59is W or R;X60is I or V;X62is K or Q;X68is selected from the group consisting of H, T, and K;X69is T or V;X70is E or T;X71is T or I; andX72is selected from the group consisting of Q, D, and E.

[0065] In some embodiments, the anti-avb6 integrin engineered polypeptide has a sequence selected from the group consisting of:TKCVVRFVFRGDLAELMLRAVKDHLKKEGPHWNITSTNNGKELVVRGIHESDAKRIAKWVEKRFPRVHTETQCD (SEQ ID NO: 1)TKCVVRFVFRGDLAELMLRAVKDHLKKEGPHWEITSTNNGKELVVRGIHESDAKRIAKWVEKRFPRVHTETQCD (SEQ ID NO: 2);TKCVVRFVFRGDLAELMLRAVKDHLKQEGPHWEITSTNNGKELVVRGIHESDAQRIAEWVEQRFPRVHTETQCD (SEQ ID NO: 3);TKCVVRFVFRGDLAELMLRAVKDHLEQEGPHWEITSTNNGKELVVRGIHESDAKRIAEWVEQRFPRVHTETQCD (SEQ ID NO: 4);TKCVVRFVFRGDLAELMLRAVKDHLEQEGPHWEITSTNNGKELVVQGIHESDAQRIAEWVEQRFPRVHTETQCD (SEQ ID NO: 5);TQCVVRFVFRGDLAELMLRAVKDHLEQEGPHWEITSTNNGKELVVEGIHESDA QRIAEWVEQRFPRVHTETQCD (SEQ ID NO: 6);DDCVVTFEFRGDTAELELRAVYEEILEENPDYEVSRTNDGRQLVVRGISREDALRWI<EWIEI<RFPRVI<VTIECD (SEQ ID NO: 7);NLCTVTFRFRGDTAELELRAVYEEILELYPDWEIERTNNGRELVVRNISEEEAEELKERIEKRFPRVTVEIDCD (SEQ ID NO: 8); andWSGR Docket No. 70774-709.601MLCTVTFRFRGDEAELQLRAAI<DYIQELYPDADVEETNDGI<ELVVRNIDAEI<A EELKEWVEKRFPRVTVEIECD (SEQ ID NO: 9).

[0066] In some embodiments, the anti-avb6 integrin engineered comprises a sequence with less than 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO. 1.

[0067] In some embodiments, anti-avb6 integrin engineered polypeptide comprises at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9.

[0068] In some embodiments, anti-avb6 integrin engineered polypeptide comprises at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9.

[0069] In some embodiments, anti-avb6 integrin engineered polypeptide comprises at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9.

[0070] In some embodiments, anti-avb6 integrin engineered polypeptide comprises at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9.

[0071] In some embodiments, anti-avb6 integrin engineered polypeptide comprises any one of SEQ ID NOs: 2-9.

[0072] In some embodiments, the anti-avb6 integrin engineered polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 3-9.

[0073] In some embodiments, the anti-avb6 integrin engineered polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 5-9.

[0074] In some embodiments, the anti-avb6 integrin engineered polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 7-9.

[0075] In some embodiments, the anti-avb6 integrin engineered polypeptide has a sequence of SEQ ID NO: 5 or 6.

[0076] In some embodiments, the anti-avb6 integrin engineered polypeptide selectively binds avb6 integrin with low nanomolar to picomolar affinity. For example, the anti-avb6 integrin engineered polypeptide may bind avb6 integrin with a KD of less than 10 nM, less than 5 nM, less than 1 nM, or less than 0.5 nM. In some embodiments, the anti-avb6 integrin engineered polypeptide binds avb6 integrin with a KD of less than 0.45 nM. In some embodiments, the anti- avb6 integrin engineered polypeptide binds avb6 integrin with a KD of less than 0.25 nM. In some embodiments, the anti-avb6 integrin engineered polypeptide binds avb6 integrin with a KD of about 0.01 nM to about 0.25 nM. In some embodiments, the anti-avb6 integrin engineeredWSGR Docket No. 70774-709.601 polypeptide binds avb6 integrin with a KD of about 0.01 nM to about 0.20 nM, about 0.01 nM to about 0.18 nM, or about 0.02 nM to about 0.16 nM.

[0077] In some embodiments, the anti-avb6 integrin engineered polypeptide exhibits the same or substantially similar affinity to avb6 integrin as the anti-avb6 integrin engineered polypeptide.

[0078] In some embodiments, the anti-avb6 integrin engineered polypeptide further comprises one or more functional domains. Non-limiting examples of functional domains include a targeting domain, a cleavable tag, a secretion signal, and an Fc domain. In some embodiments, the anti-avb6 integrin engineered polypeptide comprises a cleavable tag. In some embodiments, the anti-avb6 integrin engineered polypeptide comprises an Fc domain. When the anti-avb6 integrin engineered polypeptide is comprised of an anti-avb6 integrin engineered polypeptide and an Fc domain, the anti-avb6 integrin engineered polypeptide may also be referred to herein as a fusion protein.

[0079] In some embodiments, the sequence identity is determined by a BLASTP, CLUSTALW, MUSCLE, MAFFT algorithm, or a CLUSTALW algorithm with the Smith- Waterman homology search algorithm parameters.Fc Domains

[0080] Disclosed herein in some embodiments is a fusion protein comprising a fragment crystallizable (Fc) domain.

[0081] In some embodiments, an Fc domain comprises a variant Fc domain that comprises one or more mutations relative to a wild-type Fc domain. In some embodiments, the Fc domain is a modified domain. In some embodiments, the modified Fc domain comprises a YTE mutation compared with a wild type Fc domain.

[0082] In some embodiments, an Fc domain comprises M252Y, S254T, and T256E mutations (YTE) according to EU numbering.

[0083] In some embodiments, the Fc domain is an immunoglobulin G (IgG) Fc domain. In some embodiments, an Fc domain comprises an IgGl Fc domain. In some embodiments, an Fc domain comprises a human IgGl Fc domain.

[0084] In some embodiments, the Fc domain comprises a sequence selected from the group consisting of:WSGR Docket No. 70774-709.601EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G (SEQ ID NO: 10);EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G (SEQ ID NO: 11);EPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDL GAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKT ELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTP G (SEQ ID NO: 12);EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 13);EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 14); andEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDL GAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKT ELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTP GK (SEQ ID NO: 15).

[0085] In some embodiments, the Fc domain comprises a sequence comprising at 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-15.WSGR Docket No. 70774-709.601

[0086] In some embodiments, the Fc domain comprises a sequence selected from the group consisting of SEQ ID NOs: 10-15.Fusion Proteins

[0087] Accordingly, the present technology also provides fusion proteins comprising an anti-avb6 integrin engineered polypeptide conjugated to an Fc domain.

[0088] In some embodiments, the fusion protein comprises a) an anti-avb6 integrin engineered polypeptide as described herein, and b) an Fc domain.

[0089] In some embodiments, the Fc domain is conjugated to the N-terminus of the anti- avb6 integrin engineered polypeptide and comprises a sequence selected from SEQ ID NOs: 13- 15.

[0090] In some embodiments, the Fc domain is conjugated to the C-terminus of the anti- avb6 integrin engineered polypeptide and comprises a sequence selected from SEQ ID NOs: 10- 12.

[0091] In some embodiments, the Fc domain comprises a sequence comprising at 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-15.

[0092] In some embodiments, the Fc domain comprises a sequence selected from the group consisting of SEQ ID NOs: 10-15.

[0093] In some embodiments, the engineered polypeptide or fusion protein includes a signal polypeptide. As will be appreciated, signal polypeptides are short sequences present at the N- terminus of proteins that controls secretion of proteins in the endomembrane system, i.e., the endoplasmic reticulum. Accordingly, the engineered polypeptides and fusion proteins of the present technology may include an N-terminal signal polypeptide to facilitate delivery of the protein to the endoplasmic reticulum. In some embodiments, when the Fc domain is conjugated to the N-terminus of the avb6 integrin binding polypeptide, then the signal polypeptide is conjugated to the N-terminus of the Fc domain. In other embodiments, when the Fc domain is conjugated to the C-terminus of the anti-avb6 integrin engineered polypeptide, then the signal polypeptide is conjugated to the N-terminus of the anti-avb6 integrin engineered polypeptide.

[0094] Any suitable signal polypeptide may be included at the N-terminus of the engineered polypeptides and fusion proteins of the present technology. In some embodiments, the signalWSGR Docket No. 70774-709.601 polypeptide has a length of 14 to 20 amino acids. In some embodiments, the signal polypeptide comprises the sequence: MARAWIFFLLCLAGRALA (SEQ ID NO: 16).

[0095] In some embodiments, the Fc domain is conjugated to the N-terminus of the anti- avb6 integrin engineered polypeptide directly. Alternatively, in some embodiments, the Fc domain is conjugated to the N-terminus of the anti-avb6 integrin engineered polypeptide via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is a GS-rich peptide linker. In some embodiments, the peptide linker is GGGGS or GGSG. In some embodiments, the fusion protein comprises a sequence selected from the group consisting of:MARAWIFFLLCLAGRALAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSTKCVVRFVFRGDLAELMLRAVKDHLKKEGPH WNITSTNNGI<ELVVRGIHESDAI<RIAI<WVEI<RFPRVHTETQCD (SEQ ID NO: 17);MARAWIFFLLCLAGRALAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYI TREPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSTKCVVRFVFRGDLAELMLRAVKDHLKKEGPH WNITSTNNGI<ELVVRGIHESDAI<RIAI<WVEI<RFPRVHTETQCD (SEQ ID NO: 18);MARAWIFFLLCLAGRALAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYI TREPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSTKCVVRFVFRGDLAELMLRAVKDHLKQEGPH WEITSTNNGKELVVRGIHESDAQRIAEWVEQRFPRVHTETQCD (SEQ ID NO: 19);MARAWIFFLLCLAGRALAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYI TREPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSTKCVVRFVFRGDLAELMLRAVKDHLEQEGPHW EITSTNNGKELVVRGIHESDAKRIAEWVEQRFPRVHTETQCD (SEQ ID NO: 20);MARAWIFFLLCLAGRALAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYI TREPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHWSGR Docket No. 70774-709.601QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSTKCVVRFVFRGDLAELMLRAVKDHLEQEGPHW EITSTNNGKELVVQGIHESDAQRIAEWVEQRFPRVHTETQCD (SEQ ID NO: 21);MARAWIFFLLCLAGRALAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSTQCVVRFVFRGDLAELMLRAVKDHLEQEGPHW EITSTNNGKELVVEGIHESDAQRIAEWVEQRFPRVHTETQCD (SEQ ID NO: 22);MARAWIFFLLCLAGRALAEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQ DWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCS VVHEGLHNHHTTKSFSRTPGKGGGGSTKCVVRFVFRGDLAELMLRAVKDHLEQEGPH WEITSTNNGKELVVQGIHESDAQRIAEWVEQRFPRVHTETQCD (SEQ ID NO: 23);MARAWIFFLLCLAGRALAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGGGSGDDCVVTFEFRGDTAELELRAVYEEILEENPDYEVSR TNDGRQLVVRGISREDALRWI<EWIEI<RFPRVI<VTIECD (SEQ ID NO: 24);MARAWIFFLLCLAGRALAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGGGSGNLCTVTFRFRGDTAELELRAVYEEILELYPDWEIER TNNGRELVVRNISEEEAEELKERIEKRFPRVTVEIDCD (SEQ ID NO: 25); andMARAWIFFLLCLAGRALAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGGGSGMLCTVTFRFRGDEAELQLRAAKDYIQELYPDADVE ETNDGI<ELVVRNIDAEI<AEELI<EWVEI<RFPRVTVEIECD (SEQ ID NO: 26).WSGR Docket No. 70774-709.601

[0096] In some embodiments, the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 17-26.

[0097] In some embodiments, the Fc domain is conjugated to the C-terminus of the anti- avb6 integrin engineered polypeptide directly. Alternatively, in some embodiments, the Fc domain is conjugated to the C-terminus of the anti-avb6 integrin engineered polypeptide via a linker. In some embodiments the linker is a peptide linker. In some embodiments, the peptide linker is a G- rich or GS-rich peptide linker. In some embodiments, the peptide linker has a sequence selected from: G, GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGG. In some embodiments, the fusion protein comprises a sequence selected from the group consisting of:MARAWIFFLLCLAGRALATQCVVRFVFRGDLAELMLRAVKDHLEQEGPHWEIT STNNGKELVVEGIHESDAQRIAEWVEQRFPRVHTETQCDGGGGSGGGGSEPKSSDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CT<VSNI<ALPAPIEI<TISI<AI<G QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 27);MARAWIFFLLCLAGRALATQCVVRFVFRGDLAELMLRAVKDHLEQEGPHWEIT STNNGKELVVEGIHESDAQRIAEWVEQRFPRVHTETQCDGGGGSGGGGSGGGGEPKSS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPIEI<TIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 28);MARAWIFFLLCLAGRALATQCVVRFVFRGDLAELMLRAVKDHLEQEGPHWEIT STNNGKELVVEGIHESDAQRIAEWVEQRFPRVHTETQCDGGGGSEPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 29); andMARAWIFFLLCLAGRALATQCVVRFVFRGDLAELMLRAVKDHLEQEGPHWEIT STNNGKELVVEGIHESDAQRIAEWVEQRFPRVHTETQCDGEPKSSDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 30).WSGR Docket No. 70774-709.601

[0098] In some embodiments, the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 27-30.

[0099] As each Fc domain has an N- and C-terminus that may be conjugated to an anti-avb6 integrin engineered polypeptide, the fusion protein may comprise more than one anti-avb6 integrin engineered polypeptide. In some embodiments, the fusion protein comprises two anti-avb6 integrin engineered polypeptides. In some embodiments, an anti-avb6 integrin engineered polypeptide is conjugated to the N-terminus of the Fc domain and a second anti-avb6 integrin engineered polypeptide is conjugated to the C-terminus of the Fc domain. In some embodiments, the first and second anti-avb6 integrin engineered polypeptides are identical. In some embodiments, each of the first and second anti-avb6 integrin engineered polypeptides are conjugated to the Fc domain directly. In other embodiments, each of the first and second anti- avb6 integrin engineered polypeptides are conjugated to the Fc domain via a first and second linker, respectively. In some embodiments, the first and second linker are peptide linkers, such as, for example, GS-rich peptide linkers. In some embodiments, the first and second linkers are identical. In some embodiments, the first and second linkers are GGGGS. In some embodiments, the fusion protein comprises a sequence of:MARAWIFFLLCLAGRALATQCVVRFVFRGDLAELMLRAVKDHLEQEGPHWEIT STNNGKELVVEGIHESDAQRIAEWVEQRFPRVHTETQCDGGGGSEPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSTQCVVRFVFRGD LAELMLRAVKDHLEQEGPHWEITSTNNGKELVVEGIHESDAQRIAEWVEQRFPRVHTE TQCDG (SEQ ID NO: 31).

[0100] In some embodiments, the fusion protein comprises the sequence of SEQ ID NOs: 31.

[0101] In some embodiments, the fusion protein comprises at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31.

[0102] In some embodiments, the fusion protein comprises at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31.

[0103] In some embodiments, the fusion protein comprises at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31.WSGR Docket No. 70774-709.601

[0104] In some embodiments, the fusion protein comprises at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31.

[0105] In some embodiments, the fusion protein comprises a sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 17-22, and 24-36.

[0106] In some embodiments, the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 17-22, and 24-36.

[0107] In some embodiments, the fusion protein comprises a sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 21.

[0108] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 21.

[0109] As will be appreciated, Fc domains may exist in a monomeric form or a dimeric form. Accordingly, fusion proteins of the present technology may include a monomeric Fc domain or a dimeric Fc domain. . In some embodiments, the fusion protein includes a dimeric Fc domain. As such, the Fc domain may have four conjugation sites, one at the N- and C-terminus of each monomer. Thus, in some embodiments, the fusion protein comprises one, two, three, or four anti- avb6 integrin engineered polypeptides, each conjugated to a separate terminus of a dimeric Fc domain. For example, if the Fc domain is a dimeric Fc domain and the fusion protein comprises one anti-avb6 integrin engineered polypeptide, the anti-avb6 integrin engineered polypeptide may be conjugated to any terminus of either monomer of the Fc domain. If the fusion protein comprises two anti-avb6 integrin engineered polypeptides, then a first anti-avb6 integrin engineered polypeptide may be conjugated to the N- or C-terminus of a first monomer, and a second anti- avb6 integrin engineered polypeptide may be conjugated to the N- or C-terminus of a second monomer or the unconjugated terminus of the first monomer. If the fusion protein comprises three anti-avb6 integrin engineered polypeptides, then a first anti-avb6 integrin engineered polypeptide may be conjugated to the N-terminus of the first monomer, a second anti-avb6 integrin engineered polypeptide maybe conjugated to the C-terminus of the first monomer, and a third anti-avb6 integrin engineered polypeptide may be conjugated to the N- or C-terminus of the second monomer. If the fusion protein comprises four anti-avb6 integrin engineered polypeptides, then a first anti-avb6 integrin engineered polypeptide may be conjugated to the N-terminus of a firstWSGR Docket No. 70774-709.601 monomer of the Fc domain, a second anti-avb6 integrin engineered polypeptide may be conjugated to the C-terminus of the first monomer of the Fc domain, a third anti-avb6 integrin engineered polypeptide may be conjugated to the N-terminus of a second monomer of the Fc domain, and a fourth anti-avb6 integrin engineered polypeptide may be conjugated to the C- terminus of the second monomer of the Fc domain.

[0110] In some embodiments, when the Fc domain is a dimeric Fc domain, the fusion protein comprises a first anti-avb6 integrin engineered polypeptide conjugated to a first monomer of the Fc domain and a second anti-avb6 integrin engineered polypeptide conjugated to a second monomer of the Fc domain. In some embodiments, the first and second anti-avb6 integrin engineered polypeptides are conjugated to the first and second monomers of the Fc domain at the same terminus. For example, in such embodiments, when the first anti-avb6 integrin engineered polypeptide is conjugated to the N-terminus of the first monomer, then the second anti-avb6 integrin engineered polypeptide is conjugated to the N-terminus of the second monomer and when the first anti-avb6 integrin engineered polypeptide is conjugated to the C-terminus of the first monomer, then the second anti-avb6 integrin engineered polypeptide is conjugated to the C- terminus of the second monomer. Alternatively, in some embodiments, the first and second anti- avb6 integrin engineered polypeptides are conjugated to the first and second monomers of the Fc domain at different termini. For example, in such embodiments, when the first anti-avb6 integrin engineered polypeptide is conjugated to the N-terminus of the first monomer, then the second anti- avb6 integrin engineered polypeptide is conjugated to the C-terminus of the second monomer and when the first anti-avb6 integrin engineered polypeptide is conjugated to the C-terminus of the first monomer, then the second anti-avb6 integrin engineered polypeptide is conjugated to the N- terminus of the second monomer.[OHl] In some embodiments, the Fc domain is a homodimer. In further embodiments, when the Fc domain is a homodimer, then a first anti-avb6 integrin engineered polypeptide is conjugated to a first monomer of the Fc domain, a second anti-avb6 integrin engineered polypeptide is conjugated to a second monomer of the Fc domain, and the first and second anti-avb6 integrin engineered polypeptides are identical. In further embodiments, the first and second anti-avb6 integrin engineered polypeptides are conjugated to the same terminus of the first and second monomers of the Fc domain. In still further embodiments, the fusion protein comprises a homodimer of any one of the sequences selected from the group consisting of SEQ ID NOs: 17- 30.WSGR Docket No. 70774-709.601

[0112] Alternatively, in some embodiments, when the Fc domain is a homodimer, then a first anti-avb6 integrin engineered polypeptide is conjugated to the N-terminus of a first monomer of the Fc domain, a second anti-avb6 integrin engineered polypeptide is conjugated to the C- terminus of the first monomer of the Fc domain, a third anti-avb6 integrin engineered polypeptide is conjugated to the N-terminus of a second monomer of the Fc domain, a fourth anti-avb6 integrin engineered polypeptide is conjugated to the C-terminus of the second monomer of the Fc domain, and the first, second, third, and fourth anti-avb6 integrin engineered polypeptides are identical. In further embodiments, the fusion protein comprises a homodimer of the sequence having SEQ ID NO: 31.

[0113] Conjugation of the anti-avb6 integrin engineered polypeptides to Fc domains in the engineered polypeptides and fusion proteins of the present technology may provide the polypeptides and proteins with extended activity in vivo, relative to a control avb6 integrin binding polypeptide. For example, the fusion proteins of the present technology may exhibit one or more improved properties responsible for, or associated with, in vivo activity of anti-avb6 integrin polypeptides, relative to a control avb6 integrin binding polypeptide. In some embodiments, the fusion proteins exhibit reduced clearance in plasma, relative to a control avb6 integrin binding polypeptide. In some embodiments, the fusion proteins exhibit increased AUC in plasma, relative to a control avb6 integrin binding polypeptide. In some embodiments, the fusion proteins exhibit increased half-life in plasma relative to a control avb6 integrin binding polypeptide.

[0114] In some embodiments, the engineered polypeptides and fusion proteins exhibit one or more of the following, relative to a control avb6 integrin engineered polypeptide:(a) an increase in half-life by at least about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1,000%%, about 2,000%, about 3,000% about 4,000%, about 5,000%, about 6,000%, about 7,000%, about 8,000%, about 9,000%, about 10,000%, about 11,000%, about 12,000%, about 13,000%, about 14,000%, or about 15,000%;(b) a reduction in clearance by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%; and(c) an increase in an AUC by at least about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1,000%%, about 2,000%, about 3,000% about 4,000%, about 5,000%, about 6,000%, about 7,000%, about 8,000%, about 9,000%, about 10,000%, about 11,000%, about 12,000%, about 13,000%, about 14,000%, or about 15,000%%.WSGR Docket No. 70774-709.601

[0115] In some embodiments, the engineered polypeptides and fusion proteins exhibit one or more of the following, relative to a control avb6 integrin engineered polypeptide:(a) an increase in half-life by at least 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1,000%%, 2,000%, 3,000% 4,000%, 5,000%, 6,000%, 7,000%, 8,000%, 9,000%, 10,000%, 11,000%, 12,000%, 13,000%, 14,000%, or 15,000%;(b) a reduction in clearance by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%; and(c) an increase in an AUC by at least 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1,000%, , 2,000%, 3,000% 4,000%, 5,000%, 6,000%, 7,000%, 8,000%, 9,000%, 10,000%, 11,000%, 12,000%, 13,000%, 14,000%, or 15,000%.

[0116] The engineered polypeptides and fusion proteins of the present technology may have a clearance in plasma of less than about 150 mL / hr / kg. In some embodiments, the engineered polypeptides and fusion proteins of the present technology have a clearance in plasma of less than about 150 mL / hr / kg, less than about 100 mL / hr / kg, less than about 50 mL / hr / kg, less than about 40 mL / hr / kg, less than about 30 mL / hr / kg, less than about 20 mL / hr / kg, less than about 10 mL / hr / kg, less than about 7.5 mL / hr / kg, less than about 5 mL / hr / kg, less than about 2.5 mL / hr / kg, less than about 1 mL / hr / kg, or less than about 0.75 mL / hr / kg.

[0117] The engineered polypeptides and fusion proteins of the present technology may have a clearance in plasma of less than 150 mL / hr / kg. In some embodiments, the engineered polypeptides and fusion proteins of the present technology have a clearance in plasma of less than 150 mL / hr / kg, less than 100 mL / hr / kg, less than 50 mL / hr / kg, less than 40 mL / hr / kg, less than 30 mL / hr / kg, less than 20 mL / hr / kg, less than 10 mL / hr / kg, less than 7.5 mL / hr / kg, less than 5 mL / hr / kg, less than 2.5 mL / hr / kg, less than 1 mL / hr / kg, or less than 0.75 mL / hr / kg.

[0118] The engineered polypeptides and fusion proteins of the present technology may have reduced clearance in plasma as characterized by a threshold AUC value. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least about 50,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least about 100,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least about 200,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least about 300,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least about 400,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least about 500,000 h*ng / mL. In some embodiments, the engineered polypeptidesWSGR Docket No. 70774-709.601 and fusion proteins have an AUC of at least about 600,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least about 700,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least about 800,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least about 900,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least about 1,000,000 h*ng / mL.

[0119] In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least 50,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least 100,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least 200,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least 300,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least 400,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least 500,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least 600,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least 700,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least 800,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least 900,000 h*ng / mL. In some embodiments, the engineered polypeptides and fusion proteins have an AUC of at least 1,000,000 h*ng / mL.

[0120] Additionally, the engineered polypeptides and fusion proteins of the present technology may have reduced clearance in plasma as characterized by a threshold half-life. In some embodiments, the engineered polypeptides and fusion proteins have half-life of at least about 15 hours. In some embodiments, the engineered polypeptides and fusion proteins have half-life of at least about 20 hours. In some embodiments, the engineered polypeptides and fusion proteins have half-life of at least about 25 hours. In some embodiments, the engineered polypeptides and fusion proteins have half-life of at least about 30 hours. In some embodiments, the engineered polypeptides and fusion proteins have half-life of at least about 35 hours. In some embodiments, the engineered polypeptides and fusion proteins have half-life of at least about 40 hours.

[0121] In some embodiments, the engineered polypeptides and fusion proteins have halflife of at least 15 hours. In some embodiments, the engineered polypeptides and fusion proteins have half-life of at least 20 hours. In some embodiments, the engineered polypeptides and fusion proteins have half-life of at least 25 hours. In some embodiments, the engineered polypeptidesWSGR Docket No. 70774-709.601 and fusion proteins have half-life of at least 30 hours. In some embodiments, the engineered polypeptides and fusion proteins have half-life of at least 35 hours. In some embodiments, the engineered polypeptides and fusion proteins have half-life of at least 40 hours.

[0122] In some embodiments, the sequence identity is determined by a BLASTP, CLUSTALW, MUSCLE, MAFFT algorithm, or a CLUSTALW algorithm with the Smith- Waterman homology search algorithm parameters.Tagged Anti-avb6 Integrin Engineered Polypeptides

[0123] In some embodiments, the anti-avb6 integrin engineered polypeptide further includes a cleavable tag. A cleavable tag may be included in the anti-avb6 integrin engineered polypeptide to facilitate purification of the polypeptide and / or to detect protein-protein interactions. In some embodiments, the cleavable tag is conjugates to the N-terminus of the avb6 integrin binding polypeptide. In some embodiments, the cleavable tag includes a cleavage site and a histidine tag. In some embodiments, the histidine tag comprises the sequence HHHHHH. In some embodiments, the cleavage site comprises the sequence ENLYFQ.

[0124] The cleavable tag may be conjugated to the N-terminus of the anti-avb6 integrin engineered polypeptide via a linker. In some embodiments, the linker is a peptide linker. In further embodiments, the linker is a GS-rich peptide linker. In still further embodiments, the linker is GGGGS.

[0125] In some embodiments, the anti-avb6 integrin engineered polypeptide comprises a sequence selected from the group consisting of:HHHHHHENLYFQGGGSTKCVVRFVFRGDLAELMLRAVKDHLKKEGPHWEITST NNGKELVVRGIHESDAKRIAKWVEKRFPRVHTETQCD (LILA-0138; SEQ ID NO: 32);HHHHHHENLYFQGGGSTKCVVRFVFRGDLAELMLRAVKDHLKQEGPHWEITST NNGKELVVRGIHESDAQRIAEWVEQRFPRVHTETQCD (LILA-0139; SEQ ID NO: 33);HHHHHHENLYFQGGGSTKCVVRFVFRGDLAELMLRAVKDHLEQEGPHWEITST NNGKELVVRGIHESDAKRIAEWVEQRFPRVHTETQCD (LILA-0140; SEQ ID NO: 34);HHHHHHENLYFQGGGSTKCVVRFVFRGDLAELMLRAVKDHLEQEGPHWEITST NNGKELVVQGIHESDAQRIAEWVEQRFPRVHTETQCD (LILA-0141; SEQ ID NO: 35); andHHHHHHENLYFQGGGSTQCVVRFVFRGDLAELMLRAVKDHLEQEGPHWEITST NNGKELVVEGIHESDAQRIAEWVEQRFPRVHTETQCD (LILA-0142; SEQ ID NO: 36).Anti-avb6 Integrin Engineered Polypeptide and Fusion Protein SynthesisWSGR Docket No. 70774-709.601

[0126] The anti-avb6 integrin engineered polypeptides and fusion proteins of the present technology may be produced in host cells, such as, for example, mammalian cells like CHO and HEK293 cells, according to techniques known in the art, using transient expression or stable expression techniques. Procedures for expressing polypeptides and proteins are described generally in Gene Expression in Mammalian Cells and its Applications, Khan KH, Adv. Pharm. Bull. 3(2):257-63 (2013); the teachings of which are incorporated herein by reference. For example, the engineered polypeptide or fusion protein may be delivered to the host cell and produced by culturing the host cell under conditions for the host cell to proliferate while allowing expression of the polypeptide or protein and recovering the produced polypeptide or protein from the culture of host cells. A host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed polypolypeptide in the desired fashion. Different host cells that have specific cellular machinery and characteristic mechanisms for post- translational activities (e.g., CHO, HeLa, MDCK, HEK293, and W138) are available commercially and from the American Type Culture Collection (ATCC) and may be chosen to ensure the correct modification and processing of the expressed polypolypeptide.

[0127] A variety of expression vector and host cell systems may be used to express the engineered polypeptide or fusion protein sequences described herein. These include, but are not limited to, plasmid, or cosmid DNA expression vectors and animal cell systems. For long-term production of recombinant proteins in mammalian systems, stable expression in mammalian cell lines may be used. For example, nucleotide sequences able to encode any one of the engineered polypeptide or fusion protein sequences described herein may be transformed into cell lines using expression vectors that may contain viral origins of replication and / or endogenous expression elements and a selectable or visible marker gene on the same or on a separate vector. The present disclosure is not to be limited by the vector or host cell employed. In certain embodiments, nucleic acids able to encode engineered polypeptide or fusion protein sequences may be ligated into expression vectors. Cells may be transformed with the desired vector or vector sets.

[0128] In order to express the engineered polypeptide or fusion protein, nucleotide sequences able to encode the polypeptide or protein sequence may be inserted into an expression vector, i.e., a vector that contains the elements for transcriptional and translational control of the inserted coding sequence in a particular host. These elements may include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' un-translated regions. Suitable methods may be used to construct such expression vectors. These methods include inWSGR Docket No. 70774-709.601 vitro recombinant DNA techniques, synthetic techniques, in vivo genetic recombination and the like.

[0129] Due to the inherent degeneracy of the genetic code, DNA sequences that encode the same, substantially the same or a functionally equivalent amino acid sequence may be produced and used. The nucleotide sequences may be ordered or engineered using suitable methods in order to alter the nucleotide sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and / or expression of the gene product. Genes encoding polypeptide and protein variants may be acquired as gblock gene fragments and then cloned in a plasmid, such as, for example, pet29b, with a C-terminal tag, such as a His-tag. The genes may be cloned in pet29b between Ndel and Xhol restriction sites. Alternatively, the genes may be ordered already cloned into a plasmid, e.g., pet29b. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth. Codon-optimized nucleic acids encoding the polypeptide chains described herein are encompassed by the present disclosure.

[0130] Host cells comprising nucleotide sequences may be cultured under conditions for the transcription of the corresponding RNA (mRNA, etc.) and / or the expression and secretion of the engineered polypeptide or fusion protein from cell culture. In an exemplary embodiment, expression vectors containing nucleotide sequences able to encode the engineered polypeptide or fusion proteins of the present technology may be designed to contain signal sequences that direct secretion of the polypeptide through a eukaryotic cell membrane. Expression of the polypeptides and proteins in host cells may be carried out according to known techniques, such as but not limited to, the Studier autoinduction technique.

[0131] When using recombinant techniques, the engineered polypeptide or fusion protein can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the polypeptide or binding agent is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Cells may be lysed using a microfluidizer.

[0132] The engineered polypeptide or fusion protein of the present technology prepared from the host cells can be recovered or purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, and size exclusion chromatography. Other techniques for protein purification such as fractionation on an ion-WSGR Docket No. 70774-709.601 exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromato-focusing, SDS-PAGE, and ammonium sulfate precipitation are also available. Affinity chromatography, e.g., with a Ni-NTA column, is a common purification technique that may be used to purify the polypeptides and proteins described herein. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.

[0133] The identity of engineered polypeptides and fusion proteins produced according to techniques described above may be confirmed using mass spectrometry.Pharmaceutical Compositions

[0134] The present technology also provides pharmaceutical compositions comprising the anti-avb6 integrin engineered polypeptides or fusion proteins and one or more pharmaceutically acceptable carriers and / or excipients.

[0135] In some embodiments, the anti-avb6 integrin engineered polypeptide or fusion protein is present in a pharmaceutical composition in a therapeutically effective amount. The therapeutically effective amount may be an amount sufficient to elicit a desired response in the subject, such as, for example, inhibition of collagen and / or fibrotic tissue formation.

[0136] In some embodiments, the anti-avb6 integrin engineered polypeptide or fusion protein is present in the composition in a concentration of 0.001 mg / mL to 1000 mg / mL, relative to a total volume of the composition. For example, the anti-avb6 integrin engineered polypeptide or fusion protein is present in the composition in a concentration of 0.1 mg / mL to 500 mg / mL, 0.5 mg / mL to 250 mg / mL, 1 mg / mL to 100 mg / mL, 2.5 mg / mL to 50 mg / mL, or 5 mg / mL to 25 mg / mL, relative to a total volume of the composition. In some embodiments, the anti-avb6 integrin engineered polypeptide or fusion protein is present in the composition in a concentration of 5 mg / mL to 100 mg / mL relative to a total volume of the composition.

[0137] In some embodiments, the pharmaceutical composition comprising anti-avb6 integrin engineered polypeptide or fusion protein is formulated for intraperitoneal, intravenous, parenteral, subcutaneous, intramuscular, intracerebroventricular, intranasal, or oral administration. In some embodiments, the composition comprising the anti-avb6 integrin engineered polypeptide or fusion protein is administered to the subject parenterally. In someWSGR Docket No. 70774-709.601 embodiments, the composition comprising the anti-avb6 integrin engineered polypeptide or fusion protein is administered to the subject intravenously. In some embodiments, the composition comprising the anti-avb6 integrin engineered polypeptide or fusion protein is administered to the subject subcutaneously.

[0138] Pharmaceutical compositions disclosed herein formulated for intravenous administration may be in the form of an aqueous or non-aqueous isotonic sterile injection solution or suspension. The term “parenteral”, as used herein, includes subcutaneous, intravenous, intraperitoneal, intramuscular, and intralesional, or infusion techniques.

[0139] In some embodiments, the composition formulated for parenteral administration (e.g., intravenous or subcutaneous administration) comprises the anti-avb6 integrin engineered polypeptide or fusion protein at a concentration of about 0.001 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 100 mg / mL, 250 mg / mL, 500 mg / mL, or 1000 mg / mL, or even more, depending on the specific polypeptide selected, the desired response, the route of administration, the formulation and other factors known to those of skill in the art. In some embodiments, the composition formulated for parenteral administration (e.g., intravenous or subcutaneous administration) comprises the anti-avb6 integrin engineered polypeptide or fusion protein at a concentration of about 1 mg / mL to about 500 mg / mL. In further embodiments, the composition formulated for parenteral administration comprises the engineered polypeptide or fusion protein at a concentration of about 5 mg / mL to about 250 mg / mL, about 10 mg / mL to about 200 mg / mL, about 25 mg / mL to about 170 mg / mL or about 50 mg / mL to about 150 mg / mL.

[0140] In some embodiments, the subject is a mammal, including but not limited to a human, a non-human primate such as a chimpanzee, a domestic livestock or a farm animal such as a cow, a bison, sheep, a pig, a goat, a horse, a chicken, and a rooster, a domestic pet animal such as a dog, a cat, a rat, a mouse, and a rabbit, and a laboratory subject such as a rodent, including a rat, a mouse, and a guinea pig. In some embodiments, the subject is an animal such as a rat or a dog. In some embodiments, the subject is a human.

[0141] The pharmaceutically acceptable carriers and / or excipients may facilitate delivery (e.g., parenteral administration, in particular intravenous or subcutaneous administration) of the anti-avb6 integrin engineered polypeptides or fusion proteins to a subject. Other purposes of the pharmaceutically acceptable carriers and / or excipients include to enhance dispersion, solubility,WSGR Docket No. 70774-709.601 and stability of the anti-avb6 integrin engineered polypeptides or fusion proteins, and to reduce adverse injection site reactions.

[0142] The carriers and / or excipients of the pharmaceutical composition may generally include a pH buffered aqueous solution. In some embodiments, all components are compatible with the anti-avb6 integrin engineered polypeptides or fusion proteins (i.e., do not react or cause the engineered polypeptide or fusion protein to react) and are homogeneously dispersed or dissolved uniformly in the composition.

[0143] The pH buffered aqueous solution of the pharmaceutical compositions include water. The water may act as a diluent and include, without limitation, water for injection (WFI), sterile water, bacteriostatic water for injection (BWFI), distilled water, bidistilled water, deionized water, deionized distilled water, and reverse osmosis water. In some embodiments, the water present in the pH buffered aqueous solution is water for injection.

[0144] The pH buffered aqueous solution of the pharmaceutical composition include an organic buffer. In some embodiments, the organic buffer is selected from the group consisting of histidine, Tris, arginine, MES, ADA, PIPES, ACES, MOPSO, MOPS, Bes, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, HEPPS, TPAS, acetoamidoglycine, glycinamide, glycylglycine, tricine, and bicine. In some embodiments, the organic buffer is Tris. In some embodiments, the organic buffer is HEPES.

[0145] The buffer may be present in the pharmaceutical composition or formulation at a concentration of about 5 mM to about 100 mM. In some embodiments, the buffer is present in the pharmaceutical composition or formulation at a concentration of about 10 mM to about 100 mM, about 10 mM to about 90 mM, about 10 mM to about 80 mM, about 10 mM to about 70 mM, about 10 mM to about 6 mM, about 10 mM to about 50 mM, or about 10 mM to about 40 mM. In some embodiments, the buffer may be present in the pharmaceutical composition or formulation at a concentration of about 20 mM.

[0146] In some embodiments, the pharmaceutical composition is isotonic. In order to achieve a desirable tonicity, the composition of the present technology may further include a salt such as sodium chloride, sodium succinate, sodium sulfate, potassium chloride, magnesium chloride, magnesium sulfate, and calcium chloride. In some embodiments, the salt is sodium chloride. In some embodiments, the sodium chloride is present in the composition in a concentration of about 10 mM to about 300 mM, about 50 mM to about 250 mM, about 100 mM to about 200 mM, or about 150 mM.WSGR Docket No. 70774-709.601

[0147] In some embodiments, the pH buffered aqueous solution provides the pharmaceutical composition with a pH equivalent or close to the physiological pH levels. This may reduce adverse injection site reactions and also provide the anti-avb6 integrin engineered polypeptide or fusion protein with enhanced stability and resistance to aggregation and degradation.

[0148] In some embodiments, the composition has a pH ranging from 6.5 to 8.5. In some embodiments, the composition has a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some embodiments, the composition has a pH of about 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some embodiments, the composition has a pH ranging from about 7.4 to about 8.0.

[0149] In some embodiments, the composition has an osmolality ranging from 250 mOsm / kg to 350 mOsm / kg. For example, the composition has an osmolality ranging from 250 mOsm / kg to 360 mOsm / kg, 260 mOsm / kg to 340 mOsm / kg, 270 mOsm / kg to 330 mOsm / kg, 280 mOsm / kg to 320 mOsm / kg, 290 mOsm / kg to 310 mOsm / kg, or about 300 mOsm / kg. In some embodiments, the composition has an osmolarity of about 250 mOsm / kg, about 260 mOsm / kg, about 270 mOsm / kg, about 280 mOsm / kg, about 290 mOsm / kg, about 300 mOsm / kg, about 310 mOsm / kg, about 320 mOsm / kg, about 330 mOsm / kg, about 340 mOsm / kg, about 350 mOsm / kg, or about 360 mOsm / kg. In some embodiments, the composition has an osmolality ranging from about 275 mOsm / kg to about 330 mOsm / kg.

[0150] In some embodiments, the pharmaceutical composition further comprises additional buffer agents, preservatives, antioxidants, surfactants, and / or sweeteners.Use of Anti-avb6 Integrin Engineered Polypeptides and Fusion Proteins

[0151] The anti-avb6 integrin engineered polypeptides, fusion proteins, and pharmaceutical compositions of the present technology are useful to treat, reduce, or prevent conditions associated with aberrant avb6 integrin expression and / or activity. The anti-avb6 integrin engineered polypeptides, fusion proteins, and pharmaceutical compositions of the present technology are also useful to prevent, reduce, or mitigate collagen deposition and fibrotic tissue formation.Dosing and Administration

[0152] The pharmaceutical compositions of the present technology may be formulated to deliver a certain dose of the anti-avb6 integrin engineered polypeptide or fusion protein to a subject in need thereof. The dose may be based on the body weight of the subject. For example, in some embodiments, the pharmaceutical composition is formulated to deliver the anti-avb6WSGR Docket No. 70774-709.601 integrin engineered polypeptide or fusion protein to the subject at a dose of about 0.01 mg / kg to about 100 mg / kg, per body weight of the subject. In some embodiments, the pharmaceutical composition is formulated to deliver a dose of the anti-avb6 integrin engineered polypeptide or fusion protein of about 0.01 mg / kg to about 50 mg / kg, about 0.01 mg / kg to about 40 mg / kg, about 0.05 mg / kg to about 30 mg / kg, about 0.1 mg / kg to about 20 mg / kg, about 0.25 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 7.5 mg / kg, about 0.75 mg / kg to about 7 mg / kg, about 1 mg / kg to about 6.5 mg / kg, or about 1 mg / kg to about 6 mg / kg, per body weight of the subject. In some embodiments, the pharmaceutical composition is formulated to deliver a dose of the anti-avb6 integrin engineered polypeptide or fusion protein of about 0.01 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 7.5 mg / kg, about 0.01 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 2.5 mg / kg, about 0.01 mg / kg to about 1.5 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 7.5 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 2.5 mg / kg, or about 0.1 mg / kg to about 1.5 mg / kg, per body weight of the subj ect. In some embodiments, the pharmaceutical composition is formulated to deliver a dose of the anti-avb6 integrin engineered polypeptide or fusion protein of about 0.1 mg / kg, about 0.25 mg / kg, about 0.5 mg / kg, about 0.75 mg / kg, 1 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 12.5 mg / kg, about 15 mg / kg, about 17.5 mg / kg, about 20 mg / kg, about 22.5 mg / kg, or about 25 mg / kg, per body weight of the subject. In further embodiments, the pharmaceutical composition is formulated to deliver a dose of the anti-avb6 integrin engineered polypeptide or fusion protein of about 5 mg / kg, per body weight of the subject.

[0153] In some embodiments, the anti-avb6 integrin engineered polypeptide or fusion protein is administered to the subject once daily, twice daily, once weekly, twice weekly, three times weekly, four times weekly, five times weekly, once every two weeks, once every month, once every two months, once every three months, once every six months, or once every year.

[0154] In some embodiments, the anti-avb6 integrin engineered polypeptide or fusion protein is administered to the subject for at least about 1 day, about 1 week, about 1 month, about 3 months, about 6 months, about 1 year, or about 5 years.

[0155] In some embodiments, the anti-avb6 integrin engineered polypeptide or fusion protein is administered to the subject for at least about 1 day, about 5 days, about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 40 days, about 45 days, about 50 days, about 60 days, about 75 days, about 90 days, about 100 days, about 110 days, or about 120 days.EXAMPLESWSGR Docket No. 70774-709.601

[0156] The following examples are intended to illustrate various embodiments of the present technology. As such, the specific embodiments discussed are not to be construed as limitations on the scope of the present technology. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of present technology, and it is understood that such equivalent embodiments, are to be included herein. Further, all references cited herein are hereby incorporated by reference in their entirety, as if fully set forth herein.Anti-avB6 Engineered Polypeptides Expression and Purification

[0157] Genes encoding protein variants were ordered as gblock gene fragments from IDT and cloned in pet29b in between Ndel / Xhol restriction sites with a C-term Histag, or directly ordered from IDT already cloned into pet29b. All the mutant variants of the proteins were expressed in BL21(DE3*) using Studier autoinduction technique in standard shake flasks at 37 °C for 24 h. Cells were harvested and resuspended in 20 mM Tris, 250 mM NaCl, 20 mM Imidazole (lysis buffer). Cells were lysed using microfluidizer and cell debris was separated by centrifuging at 24,000 g for 45 min. Soluble proteins were first purified using standard Ni-NTA affinity columns followed by size exclusion chromatography (S75 10 / 300 Increase) on a GE-Akta pure FPLC system. Peak corresponding to the monomeric protein was collected and further verified by mass spectrometry.Example 2: Binding Data

[0158] The binding affinity of several exemplary anti-avb6 integrin engineered polypeptides and fusion proteins (SEQ ID NOs: 17-22 and 24-36) and a benchmark anti-avb6 integrin engineered polypeptide (SEQ ID NO: 1) to human avb6 integrin was measured by enzyme-linked immunosorbent assay (ELISA) and / or bio-layer interferometry (BLI), according to procedures known in the art. The binding affinity of one of the exemplary avb6 integrin engineered polypeptides to rat and mouse avb6 integrin was also measured by ELISA. The ELISA and BLI measurements are provided in Table 1.Table 1. Binding Affinity to avb6 IntegrinWSGR Docket No. 70774-709.601* average KD of n =2ND: not determined

[0159] As shown above, several fusion proteins comprising an anti-avb6 integrin engineered polypeptides conjugated to an Fc domain (e.g., SEQ ID NOs: 17-22 and 24-31) exhibited similar binding affinity to avb6 integrin as the benchmark anti-avb6 integrin engineered polypeptide (SEQ ID NO: 1).Example 3: Mouse Pharmacokinetic DataWild-Type Mice

[0160] To determine the pharmacokinetic parameters of the fusion proteins comprising and Fc domain of SEQ ID NO: 10 or 14 and different anti-avb6 integrin engineered polypeptides, wild-type (WT) male C57BL6J mice were intravenously administered 5 mg / kg of a fusion protein or motavizumab-YTE as a control (“control”) and plasma concentrations were measured at several time points between 0 and 100 hours post administration. Motavizumab-YTE is a monoclonal antibody with a modified Fc domain (i.e., motavizumab with amino acid substitutions M252Y / S254T / T256E [YTE]) that exhibits about 71% lower clearance and 4-fold longer half-life than unmodified motavizumab in plasma. The reduced clearance and long half-life conferred by the YTE-modified Fc domain have made motavizumab-YTE the state-of-the-art in terms of long-WSGR Docket No. 70774-709.601 acting therapeutics. As such, motavizumab-YTE was used as a control against the fusion proteins of the present technology, which contain modified or unmodified Fc domains. In some embodiments, the Fc domain is modified relative to a wild type Fc domain. In some embodiments, the Fc domain of the disclosure comprises amino acid substitutions M252Y, S254T, and T256E (YTE), according to EU numbering, relative to a wild type Fc domain. The collected data was analyzed to determine AUC, half-life (T1 / 2), and clearance (Cl) of the fusion proteins, shown in Table 2. Additionally, data collected for SEQ ID NOs: 17-22 was plotted in graphs (FIG. 1).Table 2. PK Parameters in WT Mice

[0161] As can be seen in Table 2, compared to SEQ ID NO: 18, SEQ ID NOs: 19-22 and 24-30, exhibited at least about a 28-fold increase in a plasma AUC and at least about a 30-fold reduction in clearance in WT mice.

[0162] Pharmacokinetic properties of fusion proteins comprising different Fc domains (SEQ ID NO: 11 and 12) were also evaluated. To determine the PK properties of fusion proteins comprising different Fc domains, WT mice were intravenously or subcutaneously administered 5 mg / kg of SEQ ID NO: 18, 21, and / or 22 as a benchmark, a fusion protein of SEQ ID NO: 23 or 26Y, or the control and plasma concentrations were measured at several time points between 0 and 200 hours post administration. The collected data was plotted in graphs (FIGS. 2A-2C) and analyzed to determine AUC, half-life (T1 / 2), and clearance (Cl) of the fusion proteins, shown in Table 3.WSGR Docket No. 70774-709.601Table 3. PK Parameters in WT Mice

[0163] As shown in FIGS. 2A-2C, mice that were intravenously administered 5 mg / kg SEQ ID NO: 23 and 26, had elevated plasma concentration levels of the fusion protein at 170 hours after administration, relative to the benchmark proteins.

[0164] Additionally, the PK properties of SEQ ID NO: 23 at 5 mg / kg were evaluated in WT mice via subcutaneous administration. The serum concentration of SEQ ID NO: 23 in the WT mice was collected at various time points from 0 to 200 hours after subcutaneous administration of 5 mg / kg of the fusion protein and plotted on a concentration versus time graph and compared to serum concentration of SEQ ID NO: 23 and SEQ ID NO: 21 at the same time points after intravenous administration (FIG. 3). As shown in FIG. 3, subcutaneous administration of SEQ ID NO: 23 in WT mice resulted in elevated plasma concentration of 23 at least 170 hours after administration, relative to SEQ ID NO: 21. Further, the plasma concentration of SEQ ID NO: 23 at 1700 hours after administration was similar in mice administered the fusion protein intravenously and subcutaneously.

[0165] After evaluating the PK parameters in fusion proteins comprising distinct anti-avb6 integrin engineered polypeptides, the effect of the linker used to conjugate the anti-avb6 integrin engineered polypeptide to the Fc domain on PK parameters was evaluated. In this study, fusion proteins having SEQ ID NOs: 22 and 27-30, comprising the same anti-avb6 integrin engineered polypeptide conjugated to the same Fc domain via various linkers at the N- and C-terminus were administered to WT mice at a dose of 5 mg / kg. The sequences of the linkers and conjugation terminus of the Fc domain in each fusion protein are shown in Table 4.Table 4. Sequence and Location of Linkers in anti-avb6 Engineered PolypeptidesWSGR Docket No. 70774-709.601

[0166] Plasma concentration versus time data was collected, plotted in a graph (FIG. 4A), and analyzed to determine the plasma AUC and T1 / 2 of the fusion proteins. FIGS. 4B and 4C depict graphs comparing the AUC (FIG. 4B) and T1 / 2 (FIG. 4C) values determined for each fusion protein. As shown in FIGS. 4B and 4C, the identity of the linker had an effect on the PK properties of the fusion proteins. Specifically, SEQ ID NO: 27, which had a (G4S)2 linker at the N-terminus of the Fc domain had the highest Tl / 2, at 43.6 hours, and the second highest AUC, at 2,334 hr*mg / mL.Tg32 Mice

[0167] Pharmacokinetic parameters of the fusion protein having SEQ ID NO: 21 were also evaluated in Tg32 mice, which have the mouse Fcgrt knock-out mutation and a transgene expressing the human FCGRT gene under the control of its own native promoter (hTg32). Therefore, Tg32 mice are useful in evaluating the pharmacokinetics and pharmacodynamics of human immunoglobulin G (IgG) and Fc-domain based therapeutics. In a protocol similar to that followed for the WT mice, Tg32 mice were administered 5 mg / kg of the fusion protein having SEQ ID NO: 21 or the control and plasma concentrations were measured at several time points between 0 and 200 hours post administration. The collected data was plotted in a graph (FIGS. 5 A and 5B) and analyzed to determine AUC, half-life (T1 / 2), and clearance (Cl) of the fusion proteins, shown in Table 5.Table 5. PK Parameters in Tg32 Mice

[0168] As shown in FIG. 5, SEQ ID NO: 21 was present in plasma at similar concentration as the control at 170 hours after administration in Tg32 mice. Additionally, SEQ ID NO: 21 exhibited comparable AUC and clearance in plasma compared to the control.Example 3: Cynomolsus Monkey Pharmacokinetic DataWSGR Docket No. 70774-709.601

[0169] After evaluating the PK parameters in Tg32 mice, the PK properties of SEQ ID NO: 21 in cynomolgus monkeys were evaluated. The serum concentration of SEQ ID NO: 21 in cynomolgus monkeys was collected at various time points from 0 to 600 hours after administration of 20 mg / kg of the fusion protein and plotted on a concentration versus time graph (FIG. 6). Related PK parameters including AUC, CL, and T1 / 2, were calculated based on the graphs and are provided in Table 6.Table 6. PK Parameters of SEQ ID NO: 21 in Cynomolgus Monkeys

[0170] Various embodiments of the present technology are set forth herein below in Embodiments 1-82:

[0171] Embodiment 1. An anti- alphavbeta6 (avb6) integrin engineered polypeptide comprising a sequence of Formula (IA):X1X2CX4VX6FX8FRGDX13AELX17LRAX21X22X23X24X25X26X27X28X29PX31X32X33X34X35X36TNX39GX41X42LVVX46X47IX49X50X51X52AX54X5X56X57X58X59X60EX62RFPRVX68X69X70X71X72CD (IA), wherein:X1is selected from the group consisting of T, M, N, and D;X2is selected from the group consisting of D, K, L, and Q;X4is V or T;X6is R or T;X8is selected from the group consisting of V, R, and E;X13is selected from the group consisting of L, E, and T;X17is selected from the group consisting of M, Q, and E;X21is V or A;WSGR Docket No. 70774-709.601X22is Y or K;X23is E or D;X24is selected from the group consisting of E, Y, and H;X25is selected from the group consisting of I, T, and L;X26is selected from the group consisting of K, L, Q, and E;X27is selected from the group consisting of K, E, and Q;X28is E or L;X29is selected from the group consisting of G, Y, and N;X31is H or D;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is selected from the group consisting of T, E, and S;X36is selected from the group consisting of S, E, and R;X39is N or D;X41is K or R;X42is E or Q;X46is R or Q;X47is G or N;X49is selected from the group consisting of H, D, and S;X50is selected from the group consisting of E, A, and R;X51is S or E;X52is selected from the group consisting of D, K, and E;X54is selected from the group consisting of K, E, Q, and L;X55is R or E;X56is selected from the group consisting of W, L, and I;X57is A or K;X58is K or E;X59is W or R;X60is I or V;X62is K or Q;X68is selected from the group consisting of H, T, and K;X69is T or V;X70is E or T;WSGR Docket No. 70774-709.601X71is T or I; andX72is selected from the group consisting of Q, D, and E, provided that the sequence of Formula (I) comprises less than 80% identity to the sequence: TKCVVRFVFRGDLAELMLRAVKDHLKKEGPHWNITSTNNGKELVVRGIHES DAKRIAKWVEKRFPRVHTETQCD (SEQ ID NO: 1), wherein the anti-avb6 integrin engineered polypeptide is capable of binding to avb6 integrin.

[0172] Embodiment 2. An anti-avb6 integrin engineered polypeptide comprising a sequence of Formula (IB):X1X2CX4VTFX8FRGDX13AELX17LRAX21X22X23X24IX26EX28X29PDX32X33X34X35X36TNX39GX41X42LVVRX47IX49X50EX52AX54X55X56KEX59X60EKRFPRVX68VX70IX72CD (IB), wherein:X1is selected from the group consisting of M, N, and D;X2is D or L;X4is V or T;X8is E or R;X13is E or T;X17is E or Q;X21is V or A;X22is Y or K;X23is E or D;X24is E or Y;X26is L or Q;X28is E or L;X29is N or Y;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is E or SX36is R or E;X39is N or D;X41is K or R;X42is E or Q;X47is G or N;WSGR Docket No. 70774-709.601X49is D or S;X50is selected from the group consisting of E, A, and R;X52is selected from the group consisting of D, K, and E;X54is L or E;X55is R or E;X56is W or L;X59is W or R;X60is I or V;X68is K or T;X70is E or T; andX72is E or D, provided that the sequence of Formula (I) has less than 80% identity to the sequence: TKCVVRFVFRGDLAELMLRAVKDHLKKEGPHWNITSTNNGKELVVRGIHES DAKRIAKWVEKRFPRVHTETQCD (SEQ ID NO: 1), wherein the anti-avb6 integrin engineered polypeptide is capable of binding to avb6 integrin.

[0173] Embodiment 3. An anti-avb6 integrin engineered polypeptide having a half-life greater than 25 hours in plasma, the engineered polypeptide comprising a sequence of Formula (I):X1X2CX4VX6FX8FRGDX13AELX17LRAX21X22X23X24X25X26X27X28X29PX31X32X33X34X35X36TNX39GX41X42LVVX46X47IX49X50X51X52AX54X5X56X57X58X59X60EX62RFPRVX68X69X70X71X72CD (I), wherein:X1is selected from the group consisting of T, M, N, and D;X2is selected from the group consisting of D, K, L, and Q;X4is V or T;X6is R or T;X8is selected from the group consisting of V, R, and E;X13is selected from the group consisting of L, E, and T;X17is selected from the group consisting of M, Q, and E;X21is V or A;X22is Y or K;X23is E or D;X24is selected from the group consisting of E, Y, and H;WSGR Docket No. 70774-709.601X25is selected from the group consisting of I, T, and L;X26is selected from the group consisting of K, L, Q, and E;X27is selected from the group consisting of K, E, and Q;X28is E or L;X29is selected from the group consisting of G, Y, and N;X31is H or D;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is selected from the group consisting of T, E, and S;X36is selected from the group consisting of S, E, and R;X39is N or D;X41is K or R;X42is E or Q;X46is R or Q;X47is G or N;X49is selected from the group consisting of H, D, and S;X50is selected from the group consisting of E, A, and R;X51is S or E;X52is selected from the group consisting of D, K, and E;X54is selected from the group consisting of K, E, Q, and L;X55is R or E;X56is selected from the group consisting of W, L, and I;X57is A or K;X58is K or E;X59is W or R;X60is I or V;X62is K or Q;X68is selected from the group consisting of H, T, and K;X69is T or V;X70is E or T;X71is T or I; andX72is selected from the group consisting of Q, D, and E,WSGR Docket No. 70774-709.601 provided that the sequence of Formula (I) comprises less than 80% identity to the sequence: TKCVVRFVFRGDLAELMLRAVKDHLKKEGPHWNITSTNNGKELVVRGIHES DAKRIAKWVEKRFPRVHTETQCD, (SEQ ID NO: 1), wherein the anti-avb6 integrin engineered polypeptide is capable of binding to avb6 integrin.

[0174] Embodiment 4. An anti-avb6 integrin engineered polypeptide exhibiting reduced clearance in plasma relative to a control anti-avb6 integrin polypeptide, the engineered polypeptide comprising an anti-avb6 integrin engineered polypeptide having a sequence of Formula (I):X1X2CX4VX6FX8FRGDX13AELX17LRAX21X22X23X24X25X26X27X28X29PX31X32X33X34X35X36T NX39GX41X42LVVX46X47IX49X50X51X52AX54X5X56X57X58X59X60EX62RFPRVX68X69X70X71X72CD (I), wherein:X1is selected from the group consisting of T, M, N, and D;X2is selected from the group consisting of D, K, L, and Q;X4is V or T;X6is R or T;X8is selected from the group consisting of V, R, and E;X13is selected from the group consisting of L, E, and T;X17is selected from the group consisting of M, Q, and E;X21is V or A;X22is Y or K;X23is E or D;X24is selected from the group consisting of E, Y, and H;X25is selected from the group consisting of I, T, and L;X26is selected from the group consisting of K, L, Q, and E;X27is selected from the group consisting of K, E, and Q;X28is E or L;X29is selected from the group consisting of G, Y, and N;X31is H or D;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is selected from the group consisting of T, E, and S;WSGR Docket No. 70774-709.601X36is selected from the group consisting of S, E, and R;X39is N or D;X41is K or R;X42is E or Q;X46is R or Q;X47is G or N;X49is selected from the group consisting of H, D, and S;X50is selected from the group consisting of E, A, and R;X51is S or E;X52is selected from the group consisting of D, K, and E;X54is selected from the group consisting of K, E, Q, and L;X55is R or E;X56is selected from the group consisting of W, L, and I;X57is A or K;X58is K or E;X59is W or R;X60is I or V;X62is K or Q;X68is selected from the group consisting of H, T, and K;X69is T or V;X70is E or T;X71is T or I; andX72is selected from the group consisting of Q, D, and E.

[0175] Embodiment 5. An anti-avb6 integrin engineered polypeptide exhibiting reduced clearance in plasma characterized by an AUC of at least 50,000 hr*ng / mL and a half-life in plasma greater than 25 hours, the engineered polypeptide comprising an anti-avb6 integrin engineered polypeptide having a sequence of Formula (I):X1X2CX4VX6FX8FRGDX13AELX17LRAX21X22X23X24X25X26X27X28X29PX31X32X33X34X35X36T NX39GX41X42LVVX46X47IX49X50X51X52AX54X5X56X57X58X59X60EX62RFPRVX68X69X70X71X72CD (I), wherein:X1is selected from the group consisting of T, M, N, and D;X2is selected from the group consisting of D, K, L, and Q;WSGR Docket No. 70774-709.601X4is V or T;X6is R or T;X8is selected from the group consisting of V, R, and E;X13is selected from the group consisting of L, E, and T;X17is selected from the group consisting of M, Q, and E;X21is V or A;X22is Y or K;X23is E or D;X24is selected from the group consisting of E, Y, and H; X25is selected from the group consisting of I, T, and L; X26is selected from the group consisting of K, L, Q, and E; X27is selected from the group consisting of K, E, and Q; X28is E or L;X29is selected from the group consisting of G, Y, and N;X31is H or D;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is selected from the group consisting of T, E, and S;X36is selected from the group consisting of S, E, and R;X39is N or D;X41is K or R;X42is E or Q;X46is R or Q;X47is G or N;X49is selected from the group consisting of H, D, and S;X50is selected from the group consisting of E, A, and R;X51is S or E;X52is selected from the group consisting of D, K, and E;X54is selected from the group consisting of K, E, Q, and L;X55is R or E;X56is selected from the group consisting of W, L, and I;X57is A or K;X58is K or E;WSGR Docket No. 70774-709.601X59is W or R;X60is I or V;X62is K or Q;X68is selected from the group consisting of H, T, and K;X69is T or V;X70is E or T;X71is T or I; andX72is selected from the group consisting of Q, D, and E.

[0176] Embodiment 6. The anti-avb6 integrin engineered polypeptide of any one of embodiments 1-5, wherein the anti-avb6 engineered polypeptide comprises less than 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO. 1.

[0177] Embodiment 7. The anti-avb6 integrin engineered polypeptide of any one of embodiments 1-5, comprising at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9.

[0178] Embodiment 8. The anti-avb6 integrin engineered polypeptide of any one of embodiments 1-5, comprising at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9.

[0179] Embodiment 9. The anti-avb6 integrin engineered polypeptide of any one of embodiments 1-5, comprising at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9.

[0180] Embodiment 10. The anti-avb6 integrin engineered polypeptide of any one of embodiments 1-5, comprising at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9.

[0181] Embodiment 11. The anti-avb6 integrin engineered polypeptide of any one of embodiments 1-10, comprising the amino acid sequence of any one of SEQ ID NOs: 2-9.

[0182] Embodiment 12. The anti-avb6 integrin engineered polypeptide of any one of embodiments 1-11, having a length of 74 amino acids.WSGR Docket No. 70774-709.601

[0183] Embodiment 13. The anti-avb6 integrin engineered polypeptide of any one of embodiments 1-12, wherein the polypeptide binds to human avb6 with a KD of less than 0.45 nM.

[0184] Embodiment 14. The anti-avb6 integrin engineered polypeptide of any one of embodiments 1-13, conjugated to a signal peptide.

[0185] Embodiment 15. The anti-avb6 integrin engineered polypeptide of embodiment 14, wherein the signal peptide comprises the sequence: MARAWIFFLLCLAGRALA (SEQ ID NO: 16).

[0186] Embodiment 16. The anti-avb6 integrin engineered polypeptide of any one of embodiments 14 or 15, wherein the signal peptide is conjugated to the N-terminus of the anti-avb6 integrin engineered polypeptide.

[0187] Embodiment 17. The anti-avb6 integrin engineered polypeptide of any one of embodiments 14 or 15, wherein the signal peptide is conjugated to the C-terminus of the anti-avb6 integrin binding engineered polypeptide.

[0188] Embodiment 18. The anti-avb6 integrin engineered polypeptide of any one of embodiments 1-17, further comprising a cleavable tag.

[0189] Embodiment 19. The anti-avb6 integrin engineered polypeptide of embodiment 18, wherein the cleavable tag is located at the N-terminus of the engineered polypeptide.

[0190] Embodiment 20. The anti-avb6 integrin engineered polypeptide of embodiment 18, wherein the cleavable tag is located at the C-terminus of the engineered polypeptide.

[0191] Embodiment 21. The anti-avb6 integrin engineered polypeptide of any one of embodiments 18-20, wherein the cleavable tag comprises a cleavage site and a histidine tag.

[0192] Embodiment 22. The anti-avb6 integrin engineered polypeptide of embodiment 21, wherein the histidine tag comprises the sequence HHHHHH.WSGR Docket No. 70774-709.601

[0193] Embodiment 23. The anti-avb6 integrin engineered polypeptide of embodiment 21 or 22, wherein the cleavage site comprises the sequence ENLYFQ.

[0194] Embodiment 24. The anti-avb6 integrin engineered polypeptide of any one of embodiments 14-23, wherein the signal peptide or the cleavable tag is conjugated to the anti-avb6 integrin engineered polypeptide via a linker.

[0195] Embodiment 25. The anti-avb6 integrin engineered polypeptide of embodiment 24, wherein the linker is a peptide linker.

[0196] Embodiment 26. The anti-avb6 integrin engineered polypeptide of embodiment 24 or 25, wherein the linker is a GS-rich linker.

[0197] Embodiment 27. The anti-avb6 integrin engineered polypeptide of any one of embodiments 24-26, wherein the linker is GGGGS.

[0198] Embodiment 28. The anti-avb6 integrin engineered polypeptide of any one of embodiments 18-27, wherein the engineered polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 32-36.

[0199] Embodiment 29. A fusion protein, comprising: a) the anti-avb6 integrin engineered polypeptide of any one of embodiments 1-28, and b) an Fc domain.

[0200] Embodiment 30. The fusion protein of embodiment 29, comprising at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31.

[0201] Embodiment 31. The fusion protein of any one of embodiments 29 or 30, comprising at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31.

[0202] Embodiment 32. The fusion protein of any one of embodiments 29-31, comprising at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31.WSGR Docket No. 70774-709.601

[0203] Embodiment 33. The fusion protein of any one of embodiments 29-32, comprising at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31.

[0204] Embodiment 34. The fusion protein of any one of embodiments 29-33, comprising the amino acid sequence of any one of SEQ ID NOs: 17-31.

[0205] Embodiment 35. The fusion protein of embodiment 29, wherein the fusion protein comprises a sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 17-22, and 24-36.

[0206] Embodiment 36. The fusion protein of any one of embodiments 29-35, wherein the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 17-22, and 24-36.

[0207] Embodiment 37. The fusion protein of embodiment 29, wherein the fusion protein comprises a sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 21.

[0208] Embodiment 38. The fusion protein of embodiment 29, comprising the amino acid sequence of SEQ ID NO: 21.

[0209] Embodiment 39. The fusion protein of any one of embodiments 29-38, wherein the fusion protein exhibits reduced clearance in plasma characterized by an AUC of at least 50,000 hr*ng / mL and a half-life greater than 25 hours in plasma.

[0210] Embodiment 40. The fusion protein of any one of embodiments 29-39, wherein the fusion protein exhibits reduced clearance in plasma relative to a control anti-avb6 integrin polypeptide not conjugated to the Fc domain.

[0211] Embodiment 41. The fusion protein of any one of embodiments 29-40, wherein the Fc domain is a modified Fc domain comprising one or more amino acid substitutions relative to a wild type Fc domain.WSGR Docket No. 70774-709.601

[0212] Embodiment 42. The fusion protein of embodiment 41, wherein the modified Fc domain comprises amino acid substitutions M252Y, S254T, and T256E (YTE mutation), according to EU numbering, compared with a wild type Fc domain.

[0213] Embodiment 43. The fusion protein of any one of embodiments 29-42, wherein the Fc domain is an IgG Fc domain.

[0214] Embodiment 44. The fusion protein of any one of embodiments 29-43, wherein the Fc domain comprises a sequence comprising at 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-15:

[0215] Embodiment 45. The fusion protein of any one of embodiments 29-44, wherein the Fc domain comprises a sequence selected from the group consisting of SEQ ID NOs: 10-15:

[0216] Embodiment 46. The fusion protein of any one of embodiments 29-45, further comprising a signal peptide.

[0217] Embodiment 47. The fusion protein of embodiment 46, wherein the signal peptide comprises the sequence: MARAWIFFLLCLAGRALA (SEQ ID NO: 16).

[0218] Embodiment 48. The fusion protein of embodiment 46 or 47, wherein the signal peptide is conjugated to the N-terminus of the Fc domain.

[0219] Embodiment 49. The fusion protein of embodiment 46 or 47, wherein the signal peptide is conjugated to the C-terminus of the Fc domain.

[0220] Embodiment 50. The fusion protein of any one of embodiments 46 or 47, wherein the signal peptide is conjugated to the N-terminus of the anti-avb6 integrin binding engineered polypeptide.

[0221] Embodiment 51. The fusion protein of any one of embodiments 46 or 47, wherein the signal peptide is conjugated to the C-terminus of the anti-avb6 integrin binding engineered polypeptide.WSGR Docket No. 70774-709.601

[0222] Embodiment 52. The fusion protein of any one of embodiments 46-51, wherein the anti-avb6 integrin binding engineered polypeptide is conjugated to the C-terminus of the Fc domain.

[0223] Embodiment 53. The fusion protein of any one of embodiments 46-52, wherein the anti-avb6 integrin binding engineered polypeptide is conjugated to the Fc domain via a linker.

[0224] Embodiment 54. The fusion protein of embodiment 53, wherein the linker is a peptide linker.

[0225] Embodiment 55. The fusion protein of embodiment 53 or 54, wherein the linker is a GS-rich linker.

[0226] Embodiment 56. The fusion protein of any one of embodiments 53-55, wherein the linker is GGGGS or GGSG.

[0227] Embodiment 57. The fusion protein of any one of embodiments 52-56, wherein the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 17-26.

[0228] Embodiment 58. The fusion protein of any one of embodiments 46-51, wherein the anti-avb6 integrin engineered polypeptide is conjugated to the N-terminus of the Fc domain.

[0229] Embodiment 59. The fusion protein of embodiment 58, wherein the anti-avb6 integrin engineered polypeptide is conjugated to the N-terminus of the Fc domain via a linker.

[0230] Embodiment 60. The fusion protein of embodiment 59, wherein the linker is a peptide linker.

[0231] Embodiment 61. The fusion protein of embodiment 59 or 60, wherein the linker is a G- or GS-rich linker.WSGR Docket No. 70774-709.601

[0232] Embodiment 62. The fusion protein of any one of embodiments 59-61, wherein the linker comprises a sequence selected from the group consisting of: G, GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGG.

[0233] Embodiment 63. The fusion protein of any one of embodiments 58-62, wherein the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 27-30.

[0234] Embodiment 64. The fusion protein of any one of embodiments 46-51, wherein the fusion protein comprises two or more anti-avb6 integrin binding engineered polypeptides conjugated to the Fc domain.

[0235] Embodiment 65. The fusion protein of embodiment 64, wherein one of the two or more anti-avb6 integrin binding engineered polypeptides is conjugated to the C-terminus of the Fc domain and the other of the two or more anti-avb6 integrin binding engineered polypeptides is conjugated to the N-terminus of the Fc domain.

[0236] Embodiment 66. The fusion protein of embodiments 64 or 65, wherein the two or more anti-avb6 integrin binding engineered polypeptides comprise the same sequence.

[0237] Embodiment 67. The fusion protein of any one of embodiments 64-66, wherein the two or more anti-avb6 integrin binding engineered polypeptides are conjugated to the Fc domain via a linker.

[0238] Embodiment 68. The fusion protein of embodiment 67, wherein the linker is a peptide linker.

[0239] Embodiment 69. The fusion protein of embodiments 68, wherein the peptide linker comprises a GS-rich linker.

[0240] Embodiment 70. The fusion protein of any one of embodiments 68 or 69, wherein the peptide linker is GGGGS.

[0241] Embodiment 71. The fusion protein of any one of embodiments 64-70, wherein the fusion protein comprises the sequence of SEQ ID NO: 31.WSGR Docket No. 70774-709.601

[0242] Embodiment 72. The fusion protein of any one of embodiments 29-71, wherein fusion protein is a homodimer of the Fc domain and the anti-avb6 integrin engineered polypeptide.

[0243] Embodiment 73. The fusion protein of any one of embodiments 29-72, exhibiting reduced clearance in plasma relative to a control anti-avb6 integrin engineered polypeptide or a control protein.

[0244] Embodiment 74. The fusion protein of any one of embodiments 29-73, exhibiting increased AUC in plasma relative to a control anti-avb6 integrin engineered polypeptide or a control protein.

[0245] Embodiment 75. The fusion protein of any one of embodiments 29-74, exhibiting increased half-life in plasma relative to a control anti-avb6 integrin engineered polypeptide or a control protein.

[0246] Embodiment 76. The fusion protein of any one of embodiments 29-75, exhibiting one or more of the following, relative to a control anti-avb6 integrin engineered polypeptide or a control fusion protein:(a) an increase in half-life by at least about 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1,000%, 2,000%, 3,000% 4,000%, 5,000%, 6,000%, 7,000%, 8,000%, 9,000%, 10,000%, 11,000%, 12,000%, 13,000%, 14,000%, or 15,000%;(b) a reduction in clearance by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%; and(c) an increase in an AUC by at least about 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1,000%, 2,000%, 3,000% 4,000%, 5,000%, 6,000%, 7,000%, 8,000%, 9,000%, 10,000%, 11,000%, 12,000%, 13,000%, 14,000%, or 15,000%.

[0247] Embodiment 77. The fusion protein of any one of embodiments 29-76, wherein the control protein is motavizumab-YTE.

[0248] Embodiment 78. The fusion protein of any one of embodiments 29-77, exhibiting reduced clearance in plasma characterized by an AUC of at least 50,000 h*ng / mL.WSGR Docket No. 70774-709.601

[0249] Embodiment 79. The fusion protein of any one of embodiments 29-78, exhibiting reduced clearance in plasma characterized by an AUC of at least 1,000,000 h*ng / mL.

[0250] Embodiment 80. The fusion protein of any one of embodiments 29-79, exhibiting reduced clearance in plasma characterized by a half-life of at least 25 hours.

[0251] Embodiment 81. The fusion protein of any one of embodiments 29-80, exhibiting reduced clearance in plasma characterized by a half-life of at least about 30 hours.

[0252] Embodiment 82. The anti-avb6 integrin engineered polypeptide of any one of embodiments 1-28 or the fusion protein of any one of embodiments 29-81, wherein said sequence identity is determined by a BLASTP, CLUSTALW, MUSCLE, MAFFT algorithm, or a CLUSTALW algorithm with the Smith-Waterman homology search algorithm parameters.

[0253] From the foregoing, it will be appreciated that specific embodiments of the present technology have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the present technology. Accordingly, the present technology is not limited except as by the appended claims.

Claims

1. WSGR Docket No. 70774-709.601CLAIMSWHAT IS CLAIMED IS:

1. An anti- alphavbeta6 (avb6) integrin engineered polypeptide comprising a sequence of Formula (IA): X1X2CX4VX6FX8FRGDX13AELX17LRAX21X22X23X24X25X26X27X28X29PX31X32X33X34X35X36T NX39GX41X42LVVX46X47IX49X50X51X52AX54X5X56X57X58X59X60EX62RFPRVX68X69X70X71X72CD (IA), wherein:X1is selected from the group consisting of T, M, N, and D;X2is selected from the group consisting of D, K, L, and Q;X4is V or T;X6is R or T;X8is selected from the group consisting of V, R, and E;X13is selected from the group consisting of L, E, and T;X17is selected from the group consisting of M, Q, and E;X21is V or A;X22is Y or K;X23is E or D;X24is selected from the group consisting of E, Y, and H;X25is selected from the group consisting of I, T, and L;X26is selected from the group consisting of K, L, Q, and E;X27is selected from the group consisting of K, E, and Q;X28is E or L;X29is selected from the group consisting of G, Y, and N;X31is H or D;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is selected from the group consisting of T, E, and S;X36is selected from the group consisting of S, E, and R;X39is N or D;X41is K or R;X42is E or Q;WSGR Docket No. 70774-709.601X46is R or Q;X47is G or N;X49is selected from the group consisting of H, D, and S;X50is selected from the group consisting of E, A, and R;X51is S or E;X52is selected from the group consisting of D, K, and E;X54is selected from the group consisting of K, E, Q, and L;X55is R or E;X56is selected from the group consisting of W, L, and I;X57is A or K;X58is K or E;X59is W or R;X60is I or V;X62is K or Q;X68is selected from the group consisting of H, T, and K;X69is T or V;X70is E or T;X71is T or I; andX72is selected from the group consisting of Q, D, and E, provided that the sequence of Formula (I) comprises less than 80% identity to the sequence: TKCVVRFVFRGDLAELMLRAVKDHLKKEGPHWNITSTNNGKELVVRGIHES DAKRIAKWVEKRFPRVHTETQCD (SEQ ID NO: 1), wherein the anti-avb6 integrin engineered polypeptide is capable of binding to avb6 integrin.

2. An anti-avb6 integrin engineered polypeptide comprising a sequence of Formula (IB): X1X2CX4VTFX8FRGDX13AELX17LRAX21X22X23X24IX26EX28X29PDX32X33X34X35X36TNX39G X41X42LVVRX47IX49X50EX52AX54X55X56KEX59X60EKRFPRVX68VX70IX72CD (IB), wherein:X1is selected from the group consisting of M, N, and D;X2is D or L;X4is V or T;X8is E or R;X13is E or T;WSGR Docket No. 70774-709.601X17is E or Q;X21is V or A;X22is Y or K;X23is E or D;X24is E or Y;X26is L or Q;X28is E or L;X29is N or Y;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is E or SX36is R or E;X39is N or D;X41is K or R;X42is E or Q;X47is G or N;X49is D or S;X50is selected from the group consisting of E, A, and R;X52is selected from the group consisting of D, K, and E;X54is L or E;X55is R or E;X56is W or L;X59is W or R;X60is I or V;X68is K or T;X70is E or T; andX72is E or D, provided that the sequence of Formula (I) has less than 80% identity to the sequence: TKCVVRFVFRGDLAELMLRAVKDHLKKEGPHWNITSTNNGKELVVRGIHES DAKRIAKWVEKRFPRVHTETQCD (SEQ ID NO: 1), wherein the anti-avb6 integrin engineered polypeptide is capable of binding to avb6 integrin.WSGR Docket No. 70774-709.6013. An anti-avb6 integrin engineered polypeptide having a half-life greater than 25 hours in plasma, the engineered polypeptide comprising a sequence of Formula (I): X1X2CX4VX6FX8FRGDX13AELX17LRAX21X22X23X24X25X26X27X28X29PX31X32X33X34X35X36T NX39GX41X42LVVX46X47IX49X50X51X52AX54X5X56X57X58X59X60EX62RFPRVX68X69X70X71X72CD (I), wherein:X1is selected from the group consisting of T, M, N, and D;X2is selected from the group consisting of D, K, L, and Q;X4is V or T;X6is R or T;X8is selected from the group consisting of V, R, and E;X13is selected from the group consisting of L, E, and T;X17is selected from the group consisting of M, Q, and E;X21is V or A;X22is Y or K;X23is E or D;X24is selected from the group consisting of E, Y, and H;X25is selected from the group consisting of I, T, and L;X26is selected from the group consisting of K, L, Q, and E;X27is selected from the group consisting of K, E, and Q;X28is E or L;X29is selected from the group consisting of G, Y, and N;X31is H or D;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is selected from the group consisting of T, E, and S;X36is selected from the group consisting of S, E, and R;X39is N or D;X41is K or R;X42is E or Q;X46is R or Q;X47is G or N;X49is selected from the group consisting of H, D, and S;WSGR Docket No. 70774-709.601X50is selected from the group consisting of E, A, and R;X51is S or E;X52is selected from the group consisting of D, K, and E;X54is selected from the group consisting of K, E, Q, and L;X55is R or E;X56is selected from the group consisting of W, L, and I;X57is A or K;X58is K or E;X59is W or R;X60is I or V;X62is K or Q;X68is selected from the group consisting of H, T, and K;X69is T or V;X70is E or T;X71is T or I; andX72is selected from the group consisting of Q, D, and E, provided that the sequence of Formula (I) comprises less than 80% identity to the sequence: TKCVVRFVFRGDLAELMLRAVKDHLKKEGPHWNITSTNNGKELVVRGIHES DAKRIAKWVEKRFPRVHTETQCD, (SEQ ID NO: 1), wherein the anti-avb6 integrin engineered polypeptide is capable of binding to avb6 integrin.

4. An anti-avb6 integrin engineered polypeptide exhibiting reduced clearance in plasma relative to a control anti-avb6 integrin polypeptide, the engineered polypeptide comprising an anti-avb6 integrin engineered polypeptide having a sequence of Formula (I): X1X2CX4VX6FX8FRGDX13AELX17LRAX21X22X23X24X25X26X27X28X29PX31X32X33X34X35X36T NX39GX41X42LVVX46X47IX49X50X51X52AX54X5X56X57X58X59X60EX62RFPRVX68X69X70X71X72CD (I), wherein:X1is selected from the group consisting of T, M, N, and D;X2is selected from the group consisting of D, K, L, and Q;X4is V or T;X6is R or T;X8is selected from the group consisting of V, R, and E;X13is selected from the group consisting of L, E, and T;WSGR Docket No. 70774-709.601X17is selected from the group consisting of M, Q, and E;X21is V or A;X22is Y or K;X23is E or D;X24is selected from the group consisting of E, Y, and H;X25is selected from the group consisting of I, T, and L;X26is selected from the group consisting of K, L, Q, and E;X27is selected from the group consisting of K, E, and Q;X28is E or L;X29is selected from the group consisting of G, Y, and N;X31is H or D;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is selected from the group consisting of T, E, and S;X36is selected from the group consisting of S, E, and R;X39is N or D;X41is K or R;X42is E or Q;X46is R or Q;X47is G or N;X49is selected from the group consisting of H, D, and S;X50is selected from the group consisting of E, A, and R;X51is S or E;X52is selected from the group consisting of D, K, and E;X54is selected from the group consisting of K, E, Q, and L;X55is R or E;X56is selected from the group consisting of W, L, and I;X57is A or K;X58is K or E;X59is W or R;X60is I or V;X62is K or Q;X68is selected from the group consisting of H, T, and K;WSGR Docket No. 70774-709.601X69is T or V;X70is E or T;X71is T or I; andX72is selected from the group consisting of Q, D, and E.

5. An anti-avb6 integrin engineered polypeptide exhibiting reduced clearance in plasma characterized by an AUC of at least 50,000 hr*ng / mL and a half-life in plasma greater than 25 hours, the engineered polypeptide comprising an anti-avb6 integrin engineered polypeptide having a sequence of Formula (I): X1X2CX4VX6FX8FRGDX13AELX17LRAX21X22X23X24X25X26X27X28X29PX31X32X33X34X35X36T NX39GX41X42LVVX46X47IX49X50X51X52AX54X5X56X57X58X59X60EX62RFPRVX68X69X70X71X72CD (I), wherein:X1is selected from the group consisting of T, M, N, and D;X2is selected from the group consisting of D, K, L, and Q;X4is V or T;X6is R or T;X8is selected from the group consisting of V, R, and E;X13is selected from the group consisting of L, E, and T;X17is selected from the group consisting of M, Q, and E;X21is V or A;X22is Y or K;X23is E or D;X24is selected from the group consisting of E, Y, and H;X25is selected from the group consisting of I, T, and L;X26is selected from the group consisting of K, L, Q, and E;X27is selected from the group consisting of K, E, and Q;X28is E or L;X29is selected from the group consisting of G, Y, and N;X31is H or D;X32is selected from the group consisting of W, A, and Y;X33is D or E;X34is I or V;X35is selected from the group consisting of T, E, and S;WSGR Docket No. 70774-709.601X36is selected from the group consisting of S, E, and R;X39is N or D;X41is K or R;X42is E or Q;X46is R or Q;X47is G or N;X49is selected from the group consisting of H, D, and S;X50is selected from the group consisting of E, A, and R;X51is S or E;X52is selected from the group consisting of D, K, and E;X54is selected from the group consisting of K, E, Q, and L;X55is R or E;X56is selected from the group consisting of W, L, and I;X57is A or K;X58is K or E;X59is W or R;X60is I or V;X62is K or Q;X68is selected from the group consisting of H, T, and K;X69is T or V;X70is E or T;X71is T or I; andX72is selected from the group consisting of Q, D, and E.

6. The anti-avb6 integrin engineered polypeptide of any one of claims 1-5, wherein the anti- avb6 engineered polypeptide comprises less than 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO. 1.

7. The anti-avb6 integrin engineered polypeptide of any one of claims 1-5, comprising at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9.

8. The anti-avb6 integrin engineered polypeptide of any one of claims 1-5, comprising at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9.WSGR Docket No. 70774-709.6019. The anti-avb6 integrin engineered polypeptide of any one of claims 1-5, comprising at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9.

10. The anti-avb6 integrin engineered polypeptide of any one of claims 1-5, comprising at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9.

11. The anti-avb6 integrin engineered polypeptide of any one of claims 1-10, comprising the amino acid sequence of any one of SEQ ID NOs: 2-9.

12. The anti-avb6 integrin engineered polypeptide of any one of claims 1-11, having a length of 74 amino acids.

13. The anti-avb6 integrin engineered polypeptide of any one of claims 1-12, wherein the polypeptide binds to human avb6 with a KD of less than 0.45 nM.

14. The anti-avb6 integrin engineered polypeptide of any one of claims 1-13, conjugated to a signal peptide.

15. The anti-avb6 integrin engineered polypeptide of claim 14, wherein the signal peptide comprises the sequence: MARAWIFFLLCLAGRALA (SEQ ID NO: 16).

16. The anti-avb6 integrin engineered polypeptide of any one of claims 14 or 15, wherein the signal peptide is conjugated to the N-terminus of the anti-avb6 integrin engineered polypeptide.

17. The anti-avb6 integrin engineered polypeptide of any one of claims 14 or 15, wherein the signal peptide is conjugated to the C-terminus of the anti-avb6 integrin binding engineered polypeptide.

18. The anti-avb6 integrin engineered polypeptide of any one of claims 1-17, further comprising a cleavable tag.

19. The anti-avb6 integrin engineered polypeptide of claim 18, wherein the cleavable tag is located at the N-terminus of the engineered polypeptide.WSGR Docket No. 70774-709.60120. The anti-avb6 integrin engineered polypeptide of claim 18, wherein the cleavable tag is located at the C-terminus of the engineered polypeptide.

21. The anti-avb6 integrin engineered polypeptide of any one of claims 18-20, wherein the cleavable tag comprises a cleavage site and a histidine tag.

22. The anti-avb6 integrin engineered polypeptide of claim 21, wherein the histidine tag comprises the sequence HHHHHH.

23. The anti-avb6 integrin engineered polypeptide of claim 21 or 22, wherein the cleavage site comprises the sequence ENLYFQ.

24. The anti-avb6 integrin engineered polypeptide of any one of claims 14-23, wherein the signal peptide or the cleavable tag is conjugated to the anti-avb6 integrin engineered polypeptide via a linker.

25. The anti-avb6 integrin engineered polypeptide of claim 24, wherein the linker is a peptide linker.

26. The anti-avb6 integrin engineered polypeptide of claim 24 or 25, wherein the linker is a GS-rich linker.

27. The anti-avb6 integrin engineered polypeptide of any one of claims 24-26, wherein the linker is GGGGS.

28. The anti-avb6 integrin engineered polypeptide of any one of claims 18-27, wherein the engineered polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 32-36.

29. A fusion protein, comprising: a) the anti-avb6 integrin engineered polypeptide of any one of claims 1-28, and b) an Fc domain.WSGR Docket No. 70774-709.60130. The fusion protein of claim 29, comprising at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31.

31. The fusion protein of any one of claims 29 or 30, comprising at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31.

32. The fusion protein of any one of claims 29-31, comprising at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31.

33. The fusion protein of any one of claims 29-32, comprising at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-31.

34. The fusion protein of any one of claims 29-33, comprising the amino acid sequence of any one of SEQ ID NOs: 17-31.

35. The fusion protein of claim 29, wherein the fusion protein comprises a sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 17-22, and 24-36.

36. The fusion protein of any one of claims 29-35, wherein the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 17-22, and 24-36.

37. The fusion protein of claim 29, wherein the fusion protein comprises a sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 21.

38. The fusion protein of claim 29, comprising the amino acid sequence of SEQ ID NO: 21.

39. The fusion protein of any one of claims 29-38, wherein the fusion protein exhibits reduced clearance in plasma characterized by an AUC of at least 50,000 hr*ng / mL and a half-life greater than 25 hours in plasma.WSGR Docket No. 70774-709.60140. The fusion protein of any one of claims 29-39, wherein the fusion protein exhibits reduced clearance in plasma relative to a control anti-avb6 integrin polypeptide not conjugated to the Fc domain.

41. The fusion protein of any one of claims 29-40, wherein the Fc domain is a modified Fc domain comprising one or more amino acid substitutions relative to a wild type Fc domain.

42. The fusion protein of claim 41, wherein the modified Fc domain comprises amino acid substitutions M252Y, S254T, and T256E (YTE mutation), according to EU numbering, compared with a wild type Fc domain.

43. The fusion protein of any one of claims 29-42, wherein the Fc domain is an IgG Fc domain.

44. The fusion protein of any one of claims 29-43, wherein the Fc domain comprises a sequence comprising at 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-15.

45. The fusion protein of any one of claims 29-44, wherein the Fc domain comprises a sequence selected from the group consisting of SEQ ID NOs: 10-15.

46. The fusion protein of any one of claims 29-45, further comprising a signal peptide.

47. The fusion protein of claim 46, wherein the signal peptide comprises the sequence: MARAWIFFLLCLAGRALA (SEQ ID NO: 16).

48. The fusion protein of claim 46 or 47, wherein the signal peptide is conjugated to the N- terminus of the Fc domain.

49. The fusion protein of claim 46 or 47, wherein the signal peptide is conjugated to the C- terminus of the Fc domain.

50. The fusion protein of any one of claims 46 or 47, wherein the signal peptide is conjugated to the N-terminus of the anti-avb6 integrin binding engineered polypeptide.WSGR Docket No. 70774-709.60151. The fusion protein of any one of claims 46 or 47, wherein the signal peptide is conjugated to the C-terminus of the anti-avb6 integrin binding engineered polypeptide.

52. The fusion protein of any one of claims 46-51, wherein the anti-avb6 integrin binding engineered polypeptide is conjugated to the C-terminus of the Fc domain.

53. The fusion protein of any one of claims 46-52, wherein the anti-avb6 integrin binding engineered polypeptide is conjugated to the Fc domain via a linker.

54. The fusion protein of claim 53, wherein the linker is a peptide linker.

55. The fusion protein of claim 53 or 54, wherein the linker is a GS-rich linker.

56. The fusion protein of any one of claims 53-55, wherein the linker is GGGGS or GGSG.

57. The fusion protein of any one of claims 52-56, wherein the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 17-26.

58. The fusion protein of any one of claims 46-51, wherein the anti-avb6 integrin engineered polypeptide is conjugated to the N-terminus of the Fc domain.

59. The fusion protein of claim 58, wherein the anti-avb6 integrin engineered polypeptide is conjugated to the N-terminus of the Fc domain via a linker.

60. The fusion protein of claim 59, wherein the linker is a peptide linker.

61. The fusion protein of claim 59 or 60, wherein the linker is a G- or GS-rich linker.

62. The fusion protein of any one of claims 59-61, wherein the linker comprises a sequence selected from the group consisting of: G, GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGG.WSGR Docket No. 70774-709.60163. The fusion protein of any one of claims 58-62, wherein the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 27-30.

64. The fusion protein of any one of claims 46-51, wherein the fusion protein comprises two or more anti-avb6 integrin binding engineered polypeptides conjugated to the Fc domain.

65. The fusion protein of claim 64, wherein one of the two or more anti-avb6 integrin binding engineered polypeptides is conjugated to the C-terminus of the Fc domain and the other of the two or more anti-avb6 integrin binding engineered polypeptides is conjugated to the N-terminus of the Fc domain.

66. The fusion protein of claims 64 or 65, wherein the two or more anti-avb6 integrin binding engineered polypeptides comprise the same sequence.

67. The fusion protein of any one of claims 64-66, wherein the two or more anti-avb6 integrin binding engineered polypeptides are conjugated to the Fc domain via a linker.

68. The fusion protein of claim 67, wherein the linker is a peptide linker.

69. The fusion protein of claims 68, wherein the peptide linker comprises a GS-rich linker.

70. The fusion protein of any one of claims 68 or 69, wherein the peptide linker is GGGGS.

71. The fusion protein of any one of claims 64-70, wherein the fusion protein comprises the sequence of SEQ ID NO: 31.

72. The fusion protein of any one of claims 29-71, wherein the fusion protein is a homodimer of the Fc domain and the anti-avb6 integrin engineered polypeptide.

73. The fusion protein of any one of claims 29-72, exhibiting reduced clearance in plasma relative to a control anti-avb6 integrin engineered polypeptide or a control protein.

74. The fusion protein of any one of claims 29-73, exhibiting increased AUC in plasma relative to a control anti-avb6 integrin engineered polypeptide or a control protein.WSGR Docket No. 70774-709.60175. The fusion protein of any one of claims 29-74, exhibiting increased half-life in plasma relative to a control anti-avb6 integrin engineered polypeptide or a control protein.

76. The fusion protein of any one of claims 29-75, exhibiting one or more of the following, relative to a control anti-avb6 integrin engineered polypeptide or a control fusion protein:(a) an increase in half-life by at least about 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1,000%, 2,000%, 3,000% 4,000%, 5,000%, 6,000%, 7,000%, 8,000%, 9,000%, 10,000%, 11,000%, 12,000%, 13,000%, 14,000%, or 15,000%;(b) a reduction in clearance by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%; and(c) an increase in an AUC by at least about 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1,000%, 2,000%, 3,000% 4,000%, 5,000%, 6,000%, 7,000%, 8,000%, 9,000%, 10,000%, 11,000%, 12,000%, 13,000%, 14,000%, or 15,000%.

77. The fusion protein of any one of claims 29-76, wherein the control protein is motavizumab-YTE.

78. The fusion protein of any one of claims 29-77, exhibiting reduced clearance in plasma characterized by an AUC of at least 50,000 h*ng / mL.

79. The fusion protein of any one of claims 29-78, exhibiting reduced clearance in plasma characterized by an AUC of at least 1,000,000 h*ng / mL.

80. The fusion protein of any one of claims 29-79, exhibiting reduced clearance in plasma characterized by a half-life of at least 25 hours.

81. The fusion protein of any one of claims 29-80, exhibiting reduced clearance in plasma characterized by a half-life of at least about 30 hours.

82. The anti-avb6 integrin engineered polypeptide of any one of claims 1-28 or the fusion protein of any one of claims 29-81, wherein said sequence identity is determined by a BLASTP,WSGR Docket No. 70774-709.601CLUSTALW, MUSCLE, MAFFT algorithm, or a CLUSTALW algorithm with the Smith- Waterman homology search algorithm parameters.

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