WO2023192853A2

WO2023192853A2 - Lasso peptides as endothelin b receptor antagonists

Info

Publication number: WO2023192853A2
Application number: PCT/US2023/065031
Authority: WO
Inventors: Mark J. Burk; Peter Jordan; Rajan CHAUDHARI; Matthew Lee; Gabriella COSTA MACHADO DA CRUZ; Anna LECHNER
Original assignee: Lassogen, Inc.
Priority date: 2022-03-30
Filing date: 2023-03-28
Publication date: 2023-10-05
Also published as: WO2023192853A3

Abstract

Provided herein are engineered lasso peptides that selectively bind to endothelin B receptor (ETBR), in particular ETBR1, and act as ETBR antagonists, and related compositions and methods for the management, prevention and/or treatment of an ETBR-mediated proliferative disease, such as cancer. Compositions and biosynthetic methods for producing the engineered lasso peptides are also provided.

Description

LASSO PEPTIDES AS ENDOTHELIN B RECEPTOR ANTAGONISTS

1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

63/325,465, filed March 30, 2022, the entire contents of which are incorporated by reference herein.

2. FIELD

[0002] The field of invention covers engineered lasso peptides having specificity to endothelin B receptor (ETBR), use of such engineered lasso peptides in the management, prevention or treatment of an ETBR-mediated proliferative disease, and compositions and methods of producing such engineered lasso peptides.

3. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing, which has been submitted via Patent Center. The Sequence Listing titled 200301-011002_PCT_SL.xml, which was created on March 14, 2023 and is 457,590 bytes in size, is hereby incorporated by reference in its entirety.

4. BACKGROUND

[0004] Cancer is a complex disease commonly caused by DNA damage, genetic mutations, or epigenetic modifications that support dysfunctional cellular signaling and aberrant cellular behavior leading to uncontrolled cellular growth. Over two hundred different forms of cancer are known and hundreds of drugs and drug combinations have been approved as treatments for specific cancer indications. Survival rates remain low and prevalence is increasing for many cancers (American Cancer Society. Cancer Facts & Figures 2019. Atlanta: American Cancer Society; 2019), and there exists a need for new therapeutic approaches with improved performance to combat these serious malignancies. [0005] Endothelin receptors are transmembrane G protein-coupled receptors (GPCRs) normally expressed on the surface of endothelial cells lining the inner wall of blood and lymphatic vessels. Two main receptors, endothelin receptor type A (ETAR) and endothelin receptor type B (ETBR), regulate normal vascular function by binding to one of three cognate endothelin ligands, endothelin-1 (ET-1), endothelin-2 (ET-2), or endothelin-3 (ET-3). Endothelin-induced intracellular signaling transduced by activated ETAR and ETBR controls vascular homeostasis by balancing vasoconstriction, vasodilation, angiogenesis, lymphangiogenesis, cell proliferation, and cell survival (Vignon-Zellweger, N., et al., Endothelin and endothelin receptors in the renal and cardiovascular systems, Life Sciences, 2012, 91, 490-500). ETBR activation specifically mediates the release of relaxing factors such as nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor, increases in [Ca²⁺]i, protein kinase C, mitogen-activated protein kinase, and other pathways involved in vascular contraction and cell growth (Mazzuca, M.Q., Khalil, R.A. Vascular endothelin receptor type B: structure, function and dysregulation in vascular disease, Biochem Pharmacol. 2012; 84(2): 147-162).

[0006] Endothelin receptor antagonists have been reported in the literature and have been largely studied in the context of pulmonary arterial hypertension (PAH) and other cardiovascular diseases (Aubert, J., et al., Endothelin receptor antagonists beyond pulmonary arterial hypertension, cancer and fibrosis, J. Med. Chem. 2016, 59, 8168-8188; Davenport, A.P., et al., New drugs and emerging targets in endothelin signaling pathway and prospects for precision medicine, Physiol. Res., 2018, 67 (Suppl. 1), S37-S54). For example, a modified tetrapeptide molecule BQ-788 was initially developed to characterize the physiological and pathological roles of endothelin receptors in the context of hypertension and pulmonary diseases (Ishikawa, K. et al., Biochemical and pharmacological profile of a potent and selective endothelin B-receptor antagonist, BQ-788, Proc. Nat. Acad. Sci. USA, 1994, 91,4892-4896). Most endothelin receptor antagonists are either ETAR selective or antagonize both ETAR and ETBR, and several have been approved for treating PAH and related disorders. Relatively few potent and selective ETBR antagonists have been described (Aubert, J., et al., Endothelin receptor antagonists beyond pulmonary arterial hypertension, cancer and fibrosis, J. Med. Chem. 2016, 59, 8168-8188), and no selective ETBR antagonist has been approved for commercial use. Importantly, there is also no cancer medication approved, which medication advances its anti-cancer therapeutic effect through ETBR antagonism.

[0007] Despite tremendous efforts in the field of cancer medication, cancer mortality rate remains high across the globe. Thus, there remains urgent needs for the development of effective new cancer medication. The present disclosure meets this need. 5. SUMMARY

[0008] Provided herein are engineered lasso peptides that selectively bind to endothelin B receptor (ETBR), in particular ETBR1, and act as ETBR antagonists, and related compositions and methods for the management, prevention and/or treatment of an ETBR- mediated proliferative disease, such as cancer. Compositions and biosynthetic methods for producing the engineered lasso peptides are also provided.

[0009] Particularly, in a first aspect of the present disclosure, provided herein is an engineered lasso peptide comprising a variant of amino acid sequence SEQ ID NO: 1, wherein the engineered lasso peptide comprises one or more amino acid substitutions, and wherein the engineered lasso peptide, when cyclized, has at least a 1.5-fold higher specific binding affinity to endothelin B receptor (ETBR) compared to a lasso peptide consisting of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions allows for increased hydrogen bonding of the engineered lasso peptide to residues in the pocket of ETBR that bind endothelin ligands or in the receptor capping region of ETBR.

[0010] In some embodiments, the engineered lasso peptide having at least a 1.5-fold higher specific binding affinity to ETBR comprises a F11Y substitution. In some embodiments, the engineered lasso peptide comprising a F11Y substitution further comprises a substitution selected from the group consisting of N2A, N2D, N2S, W3A, W3D, W3E, W3Y, W3H, H4A, H4I, H4Q, H4M, H4L, H4W, H4Y, G5A, T6A, T6E, T6I, T6K, T6L, T6V, T6S, T6H, S7E, S7F, S7I, S7L, S7N, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, W10A, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, W16E, and W16K. In some embodiments, the engineered lasso peptide comprising a F11Y substitution further comprises a substitution selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, and W16K.

[0011] In some embodiments, the engineered lasso peptide having at least a 1.5-fold higher specific binding affinity to ETBR comprises a F12H substitution. In some embodiments, the engineered lasso peptide comprising a F12H substitution further comprises a substitution selected from the group consisting of N2A, N2D, N2S, W3A, W3D, W3E, W3Y, W3H, H4A, H4I, H4Q, H4M, H4L, H4W, H4Y, G5A, T6A, T6E, T6I, T6K, T6L, T6V, T6S, T6H, S7E, S7F, S7I, S7L, S7N, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, W10A, F11Y, F11 S, F11T, F11W, N13S, N13F, N13H, Y15F, Y15L, Y15H, W16E, and W16K. In some embodiments, the engineered lasso peptide comprising a F12H substitution further comprises a substitution selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F11Y, F11S, F11T, N13F, N13H, Y15F, Y15L, Y15H, and W16K.

[0012] In some embodiments, the engineered lasso peptide having at least a 1.5-fold higher specific binding affinity to ETBR comprises a F12Y substitution. In some embodiments, the engineered lasso peptide comprising a F12Y substitution further comprises a substitution selected from the group consisting of N2A, N2D, N2S, W3A, W3D, W3E, W3Y, W3H, H4A, H4I, H4Q, H4M, H4L, H4W, H4Y, G5A, T6A, T6E, T6I, T6K, T6L, T6V, T6S, T6H, S7E, S7F, S7I, S7L, S7N, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, W10A, F11Y, F11 S, F11T, F11W, N13S, N13F, N13H, Y15F, Y15L, Y15H, W16E, and W16K. In some embodiments, the engineered lasso peptide comprising a F12Y substitution further comprises a substitution selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F11Y, F11S, F11T, N13F, N13H, Y15F, Y15L, Y15H, and W16K.

[0013] In some embodiments, the engineered lasso peptide having at least a 1.5-fold higher specific binding affinity to ETBR comprises substitutions selected from the group consisting of: a) H4L and F11Y; b) H4M and F11Y; c) F11Y and F12H; and d) F11Y and F12Y.

[0014] In some embodiments, the engineered lasso peptide having at least a 1.5-fold higher specific binding affinity to ETBR comprises a F11Y substitution and two amino acid substitutions selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, and W16K. In some embodiments, the engineered lasso peptide having at least a 1.5-fold higher specific binding affinity to ETBR comprises substitutions selected from the group consisting of: a) H4M, F11Y, and F12Y; b) W3E, H4M, and F11Y; c) W3Y, H4M, and F11Y; and d) W3H, H4M, andF11Y.

[0015] In some embodiments, the engineered lasso peptide having at least a 1.5-fold higher specific binding affinity to ETBR comprises a F11Y substitution and three amino acid substitutions selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, and W16K. In some embodiments, the engineered lasso peptide having at least a 1.5-fold higher specific binding affinity to ETBR comprises substitutions selected from the group consisting of: a) W3D, H4M, F11Y, and F12Y; b) W3E, H4M, F11Y, and F12Y; c) W3Y, H4M, F11Y, and F12Y; and d) W3H, H4M, F11Y, and F12Y.

[0016] In some embodiments, the engineered lasso peptide having at least a 1.5-fold higher specific binding affinity to ETBR comprises a F11Y substitution and four amino acid substitutions selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, and W16K. In some embodiments, the engineered lasso peptide having at least a 1.5-fold higher specific binding affinity to ETBR comprises substitutions selected from the group consisting of: a) W3D, H4M, F11Y, F12Y, and N13F; b) W3E, H4M, F11Y, F12Y, and N13F; c) W3Y, H4M, F11Y, F12Y, and N13F; d) W3H, H4M, F11Y, F12Y, and N13F; e) W3D, H4M, F11Y, F12Y, and N13H; f) W3E, H4M, F11Y, F12Y, and N13H; g) W3Y, H4M, F11Y, F12Y, and N13H; and h) W3H, H4M, F11Y, F12Y, and N13H.

[0017] In some embodiments, the engineered lasso peptide having at least a 1.5-fold higher specific binding affinity to ETBR comprises a F11Y substitution and five amino acid substitutions selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, and W16K. In some embodiments, the engineered lasso peptide having at least a 1.5-fold higher specific binding affinity to ETBR comprises substitutions selected from the group consisting of: a) W3D, H4M, F11Y, F12Y, N13F, and S7I; b) W3E, H4M, F11Y, F12Y, N13F, and S7I; c) W3Y, H4M, F11Y, F12Y, N13F, and S7I; d) W3H, H4M, F11Y, F12Y, N13F, and S7I; e) W3D, H4M, F11Y, F12Y, N13H, and S7I; f) W3E, H4M, F11Y, F12Y, N13H, and S7I; g) W3Y, H4M, F11Y, F12Y, N13H, and S7I; h) W3H, H4M, F11Y, F12Y, N13H, and S7I; i) W3D, H4M, F11Y, F12Y, N13F, and S7Y; j) W3E, H4M, F11Y, F12Y, N13F, and S7Y; k) W3Y, H4M, F11Y, F12Y, N13F, and S7Y; 1) W3H, H4M, F11Y, F12Y, N13F, and S7Y; m) W3D, H4M, F11Y, F12Y, N13H, and S7Y; n) W3E, H4M, F11Y, F12Y, N13H, and S7Y; o) W3Y, H4M, F11Y, F12Y, N13H, and S7Y; p) W3H, H4M, F11Y, F12Y, N13H, and S7Y; q) W3D, H4M, F11Y, F12Y, N13F, and S7F; r) W3E, H4M, F11Y, F12Y, N13F, and S7F; s) W3Y, H4M, F11Y, F12Y, N13F, and S7F; t) W3H, H4M, F11Y, F12Y, N13F, and S7F; u) W3D, H4M, F11Y, F12Y, N13H, and S7F; v) W3E, H4M, F11Y, F12Y, N13H, and S7F; w) W3Y, H4M, F11Y, F12Y, N13H, and S7F; x) W3H, H4M, F11Y, F12Y, N13H, and S7F; y) W3D, H4M, F11Y, F12Y, N13F, and S7K; z) W3E, H4M, F11Y, F12Y, N13F, and S7K; aa) W3Y, H4M, F11Y, F12Y, N13F, and S7K; bb) W3H, H4M, F11Y, F12Y, N13F, and S7K; cc) W3D, H4M, F11Y, F12Y, N13H, and S7K; dd) W3E, H4M, F11Y, F12Y, N13H, and S7K; ee) W3Y, H4M, F11Y, F12Y, N13H, and S7K; ff) W3H, H4M, F11Y, F12Y, N13H, and S7K; gg) W3D, H4M, F11Y, F12Y, N13F, and S7R; hh) W3E, H4M, F11Y, F12Y, N13F, and S7R; ii) W3Y, H4M, F11Y, F12Y, N13F, and S7R; jj) W3H, H4M, F11Y, F12Y, N13F, and S7R; kk) W3D, H4M, F11Y, F12Y, N13H, and S7R; 11) W3E, H4M, F11Y, F12Y, N13H, and S7R; mm) W3Y, H4M, F11Y, F12Y, N13H, and S7R; and nn) W3H, H4M, F11Y, F12Y, N13H, and S7R.

[0018] In a second aspect of the present disclosure, provided herein is an engineered lasso peptide comprising a variant of amino acid sequence SEQ ID NO: 1, wherein the engineered lasso peptide comprises one or more amino acid substitutions selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F11Y, F11S, F11T, Fl 2H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, and W16K. In some embodiments, the one or more amino acid substitutions comprises two, three, four, five, or six amino acid substitutions. In some embodiments, the one or more amino acid substitutions comprises two amino acid substitutions. In some embodiments, the two amino acid substitutions are selected from the group consisting of: a) H4L and F11Y; b) H4M and F11Y; c) T6P and P8F; d) T6P and P8L; e) T6V and S7N; f) S7P and P8F; g) S7P and P8L; h) F11Y and F12H; and i) F11Y and F12Y. In some embodiments, the one or more amino acid substitutions comprises three amino acid substitutions. In some embodiments, the three amino acid substitutions are selected from the group consisting of: a) H4M, F11Y, and F12Y; b) W3E, H4M, and F11Y; c) W3Y, H4M, and F11Y; and d) W3H, H4M, and F11Y. In some embodiments, the one or more amino acid substitutions comprises four amino acid substitutions. In some embodiments, the four amino acid substitutions are selected from the group consisting of: a) W3D, H4M, F11Y, and F12Y; b) W3E, H4M, F11Y, and F12Y; c) W3Y, H4M, F11Y, and F12Y; and d) W3H, H4M, F11Y, and F12Y. In some embodiments, the one or more amino acid substitutions comprises five amino acid substitutions. In some embodiments, the five amino acid substitutions are selected from the group consisting of: a) W3D, H4M, F11Y, F12Y, and N13F; b) W3E, H4M, F11Y, F12Y, and N13F; c) W3Y, H4M, F11Y, F12Y, and N13F; d) W3H, H4M, F11Y, F12Y, and N13F; e) W3D, H4M, F11Y, F12Y, and N13H; f) W3E, H4M, F11Y, F12Y, and N13H; g) W3Y, H4M, F11Y, F12Y, and N13H; and h) W3H, H4M, F11Y, F12Y, and N13H. In some embodiments, the one or more amino acid substitutions comprises six amino acid substitutions. In some embodiments, the six amino acid substitutions are selected from the group consisting of: a) W3D, H4M, F11Y, F12Y, N13F, and S7I; b) W3E, H4M, F11Y, F12Y, N13F, and S7I; c) W3Y, H4M, F11Y, F12Y, N13F, and S7I; d) W3H, H4M, F11Y, F12Y, N13F, and S7I; e) W3D, H4M, F11Y, F12Y, N13H, and S7I; f) W3E, H4M, F11Y, F12Y, N13H, and S7I; g) W3Y, H4M, F11Y, F12Y, N13H, and S7I; h) W3H, H4M, F11Y, F12Y, N13H, and S7I; i) W3D, H4M, F11Y, F12Y, N13F, and S7Y; j) W3E, H4M, F11Y, F12Y, N13F, and S7Y; k) W3Y, H4M, F11Y, F12Y, N13F, and S7Y; 1) W3H, H4M, F11Y, F12Y, N13F, and S7Y; m) W3D, H4M, F11Y, F12Y, N13H, and S7Y; n) W3E, H4M, F11Y, F12Y, N13H, and S7Y; o) W3Y, H4M, F11Y, F12Y, N13H, and S7Y; p) W3H, H4M, F11Y, F12Y, N13H, and S7Y; q) W3D, H4M, F11Y, F12Y, N13F, and S7F; r) W3E, H4M, F11Y, F12Y, N13F, and S7F; s) W3Y, H4M, F11Y, F12Y, N13F, and S7F; t) W3H, H4M, F11Y, F12Y, N13F, and S7F; u) W3D, H4M, F11Y, F12Y, N13H, and S7F; v) W3E, H4M, F11Y, F12Y, N13H, and S7F; w) W3Y, H4M, F11Y, F12Y, N13H, and S7F; x) W3H, H4M, F11Y, F12Y, N13H, and S7F; y) W3D, H4M, F11Y, F12Y, N13F, and S7K; z) W3E, H4M, F11Y, F12Y, N13F, and S7K; aa) W3Y, H4M, F11Y, F12Y, N13F, and S7K; bb) W3H, H4M, F11Y, F12Y, N13F, and S7K; cc) W3D, H4M, F11Y, F12Y, N13H, and S7K; dd) W3E, H4M, F11Y, F12Y, N13H, and S7K; ee) W3Y, H4M, F11Y, F12Y, N13H, and S7K; ff) W3H, H4M, F11Y, F12Y, N13H, and S7K; gg) W3D, H4M, F11Y, F12Y, N13F, and S7R; hh) W3E, H4M, F11Y, F12Y, N13F, and S7R; ii) W3Y, H4M, F11Y, F12Y, N13F, and S7R; jj) W3H, H4M, F11Y, F12Y, N13F, and S7R; kk) W3D, H4M, F11Y, F12Y, N13H, and S7R; 11) W3E, H4M, F11Y, F12Y, N13H, and S7R; mm) W3Y, H4M, F11Y, F12Y, N13H, and S7R; and nn) W3H, H4M, F11Y, F12Y, N13H, and S7R.

[0019] In some embodiments, the engineered lasso peptide is at least 10% more stable as measured by thermal degradation or proteolytic degradation through hydrolysis of a peptide bond compared to a lasso peptide consisting of the amino acid sequence of SEQ ID NO: 1. [0020] In some embodiments, the engineered lasso peptide is at least 10% more soluble in water or a mixture containing water compared to a lasso peptide consisting of the amino acid sequence of SEQ ID NO: 1. [0021] In a third aspect of the present disclosure, provided herein is an engineered lasso peptide comprising an amino acid sequence selected from SEQ ID NOS: 2-117.

[0022] In a fourth aspect of the present disclosure, provided herein is an engineered lasso peptide consisting of an amino acid sequence selected from SEQ ID NOS: 2-117.

[0023] In some embodiments, the engineered lasso peptide further comprises a leader sequence. Such a leader sequence, in some embodiments, comprises the amino acid sequence of MTEQSEQTPTEYIPPMLVEVGEFTEDTL (SEQ ID NO: 118).

[0024] In some embodiments, the engineered lasso peptide further comprises a C-terminal tryptophan (W) modification selected from the group consisting of: a) tryptophan having a C- terminal methyl ester group (-CO2Me) in place of the carboxylic acid group (-CO2H) (W- OMe); b) tryptophan having a C-terminal benzyl ester group (-CO2Bn) in place of the carboxylic acid group (-CO2H) (W-OBn); c) tryptophan having a C-terminal amide group (- CONH2) in place of the carboxylic acid group (-CO2H) (W-NH2); d) 7-hydroxyl-trptophan (W-7-OH); e) 2-naphthylalanine (Nal) in place of W; and f) an aza derivative of tryptophan - (2S)-2-amino-3-(lH-pyrrolo[5,4-b]pyridin-3-yl)propanoic acid - in place of W having the structure of:

(Tm).

[0025] In some embodiments, the engineered lasso peptide is G1-D9 cyclized. In some embodiments, the engineered lasso peptide competes with endothelin for the binding with ETBR. Such an endothelin, in some embodiments, is endothelin 1, endothelin 2 and/or endothelin 3.

[0026] In some embodiments, the engineered lasso peptide preferentially binds to ETBR over endothelin A receptor (ETAR). In some embodiments, the engineered lasso peptide specifically antagonizes ETBR. In some embodiments, the engineered lasso peptide preferentially binds to ETBR1 over ETBR2. In some embodiments, the engineered lasso peptide specifically antagonizes ETBR1.

SUBSTITUTE SHEET ( RULE 26) [0027] In a fifth aspect of the present disclosure, provided herein is a pharmaceutical composition comprising an engineered lasso peptide described herein (e.g., SEQ ID NOS: 2- 117) and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition of the present disclosure further comprises a second therapeutic agent for managing, preventing or treating cancer. In some embodiments, the second therapeutic agent is chemotherapy or immunotherapy for cancer. In some embodiments, the second therapeutic agent is an anti -cancer vaccine or immune checkpoint modulator. [0028] In a sixth aspect of the present disclosure, provided herein is a method for managing, preventing, or treating an endothelin B receptor (ETB Remediated proliferative disease producing neoplastic cells in a subject. Such a method can include administering to a subject a therapeutically effective amount of an engineered lasso peptide described herein (e.g., SEQ ID NOS: 2-117) or a pharmaceutical composition described herein.

[0029] In some embodiments, upon administration, the engineered lasso peptide (a) antagonizes an ETBR-mediated signaling pathway; (b) reduces ETBR levels on the surface of neoplastic cells and/or endothelial cells in the microenvironment of the neoplastic cells due to ligand-induced ETBR internalization; and/or downregulates ETBR expression on the surface of the neoplastic cells and/or endothelial cells in the microenvironment of the neoplastic cells. In some embodiments, the antagonism of the ETBR-mediated signaling pathway is measured by (a) inhibition of release of relaxing factors; (b) upregulation of intercellular adhesion molecule- 1 (ICAM-1) expression and clustering; (c) increase in migration of intraepithelial tumor infiltrating leukocytes (TILs) into the microenvironment of the neoplastic cells; (d) inhibition of angiogenesis in the microenvironment of neoplastic cells; (e) inhibition of growth and/or metastasis of neoplastic cells; and/or (f) increase in apoptosis of neoplastic cells.

[0030] In some embodiments, upon administration, the engineered lasso peptide inhibits release of relaxing factors selected from nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor, Ca²⁺, protein kinase C, mitogen-activated protein kinase, or any combination thereof.

[0031] In some embodiments, upon administration, the engineered lasso peptide increases migration of TILs into the microenvironment of the neoplastic cells, and the TILs comprise neutrophils, T cells, B cells, NK cells, monocytes or a combination thereof. In particular embodiments, the monocytes comprise macrophages and/or dendritic cells. [0032] In some embodiments, the proliferative disease being treated produces neoplastic cells expressing ETBR. In some embodiments, the subject being treated expresses ETBR in endothelial cells of vasculature in the microenvironment of the neoplastic cells. In some embodiments, the ETBR being expressed is ETBR1, ETBR2 or both ETBR1 and ETBR2. [0033] In some embodiments, the proliferative disease being treated is cancer. In some embodiments, the cancer is breast cancer, pancreatic cancer, hepatocellular cancer, prostate cancer, ovarian cancer, gastric cancer, brain or spinal cancer, melanoma, cancer of the head and neck, colorectal cancer, bladder cancer, vulvar cancer, esophageal squamous cell carcinoma, renal cancer, cervical cancer, salivary gland carcinoma, lung cancer, multiple myeloma, or Kaposi’s sarcoma. In some embodiments, the brain or spinal cancer is a glioma. In some embodiments, the glioma is a glioblastoma. In some embodiments, the proliferative disease being treated is melanoma. In some embodiments, the proliferative disease being treated is breast cancer. In some embodiments, the proliferative disease being treated is ovarian cancer.

[0034] In some embodiments, upon administration of the engineered lasso peptide, the maximal percent inhibition of the ETBR-mediated signaling pathway is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, upon administration of the engineered lasso peptide, the maximal percent reduction of ETBR levels is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, upon administration of the engineered lasso peptide, the maximal percent downregulation of ETBR expression is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0035] In some embodiments, the engineered lasso peptide is conjugated to an agent. In some embodiments, the engineered lasso peptide is conjugated to an agent selected from the group consisting of a radioisotope, a metal chelator, an enzyme, a protein, a peptide, an antibody, an antibody fragment, a nanobody, a cytotoxic compound, a fluorescent compound, a bioluminescent compound, and a chemiluminescent compound.

[0036] In some embodiments, the method further comprises co-administering to the subject a second therapeutic agent with the engineered lasso peptide. In some embodiments, the second therapeutic agent is conjugated with the engineered lasso peptide. In some embodiments, the second therapeutic agent is an immunotherapy or chemotherapy. In particular embodiments, the immunotherapy is an anti-cancer vaccine or an immune checkpoint modulator.

[0037] In a seventh aspect of the present disclosure, provided herein is a recombinant nucleic acid encoding the engineered lasso peptide described herein (e.g., SEQ ID NOS: 2- 117). In some embodiments, such a recombinant nucleic acid comprises a nucleotide sequence described herein (e.g., SEQ ID NOS: 119-235).

[0038] In an eighth aspect of the present disclosure, provided herein is a recombinant nucleic acid encoding a lasso precursor peptide of an engineered lasso peptide described herein (e.g., SEQ ID NOS: 238-363). In some embodiments, such a recombinant nucleic acid includes a nucleotide sequence encoding the precursor peptide described herein (e.g, SEQ ID NOS: 364-480). In some embodiments, a recombinant nucleic acid includes a nucleotide sequence encoding the engineered lasso peptide or precursor peptide described herein that is operatively linked to a promoter.

[0039] In a ninth aspect of the present disclosure, provided herein is a vector comprising a recombinant nucleic acid described herein.

[0040] In a tenth aspect of the present disclosure, provided herein a non-naturally occurring microbial organism having a recombinant nucleic acid described herein or a vector described herein.

[0041] In an eleventh aspect of the present disclosure, provided herein is a method for producing an engineered lasso peptide using a non-naturally occurring microbial organism described herein. In some embodiments, the method comprises introducing into the microbial organism a first nucleic acid sequence comprising a recombinant nucleic acid described herein or a vector described herein and a second nucleic acid sequence encoding a lasso peptide biosynthesis component; and culturing the microbial organism under a condition suitable for lasso formation to produce the engineered lasso peptide. In some embodiments, the first nucleic acid sequence encodes an engineered lasso peptide having a leader sequence and the lasso peptide biosynthesis component comprises a lasso peptidase capable of catalyzing removal of the leader sequence.

[0042] In some embodiments, the method for producing an engineered lasso peptide provided herein includes a lasso peptide biosynthesis component that includes a lasso cyclase capable of cyclizing a linear lasso core sequence to a mature lasso peptide.

[0043] In some embodiments, the method for producing an engineered lasso peptide provided herein includes a lasso peptidase and a lasso cyclase, wherein the method comprises introducing the second nucleic acid sequence encoding the lasso cyclase and a third nucleic acid sequence encoding the lasso peptidase.

[0044] In some embodiments, the method for producing an engineered lasso peptide provided herein includes a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE). In some embodiments, such a method includes introducing the second nucleic acid sequence encoding the lasso cyclase and a fourth nucleic acid sequence encoding the RRE.

[0045] In some embodiments, the method for producing an engineered lasso peptide provided herein includes a lasso peptidase, a lasso cyclase and a RRE. In some embodiments, such a method includes introducing the second nucleic acid sequence encoding the lasso cyclase, a third nucleic acid sequence encoding the lasso peptidase, and a fourth nucleic acid sequence encoding the RRE.

[0046] In some embodiments, at least two of the first, second, third and fourth nucleic acid sequences introduced in the methods for producing an engineered lasso peptide are in a same nucleic acid molecule.

[0047] In some embodiments, the microbial organism is E.coli, Vibrio natriegens, Burholderia spp., Corynebacterium glutamicum, or Sphingomonas subterranean, Pseudomonas fluor escens, Saccharomyces cerevisiae, Pichia pastoris, Rhodococcus jostii, Saccharopolyspora erythraea, Streptomyces lividans, Streptomyces coelicolor, Streptomyces albus, or Streptomyces venezuelae . In some embodiments, the culturing of the method for producing an engineered lasso peptide is performed under aerobic and/or glucose-limiting conditions. In some embodiments, the method further includes isolating the engineered lasso peptide from the culture medium of the microbial organism.

6. BRIEF DESCRIPTION OF THE FIGURES

[0048] The details of one or more embodiments of the subject application are set forth in the accompanying drawings and the description below. Other features, objects, and benefits of the embodiments described herein will be apparent from the description and drawings, and from the claims. All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.

[0049] The embodiments of the description described herein are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed in the following drawings or detailed description. Rather, the embodiments are chosen and described so that others skilled in the art can appreciate and understand the principles and practices of the description. [0050] FIG. 1 is a schematic illustration of a lasso peptide with the characteristic lasso (lariat) topology.

[0051] FIG. 2 shows the lasso peptide structure of SEQ ID NO 1 and the positions for single-site and multi-site mutations as described herein shown in black.

[0052] FIG. 3 shows a schematic illustration of the lasso peptide biosynthesis pathway, including the genes and gene products involved in the enzymatic reactions resulting in formation of matured lasso peptide having the characteristic lasso (lariat) topology.

[0053] FIG. 4 shows a schematic illustration of two step transformation leading to formation of lasso peptide structure of SEQ ID NO 1 through lasso peptidase cleavage of a lasso precursor peptide derived from a lasA gene, followed by lasso cyclase conversion of the core sequence. SEQ ID NO 1 structure shows the loop, ring, and tail and the positions of residues Gl, D9, and W16.

[0054] FIG. 5 shows an exemplary expression vector for lasso peptide variants of SEQ ID NO: 1. GenBank accession numbers for lasA, lasC las Bl and las B2 are indicated. Abbreviations: NitRP - NitR promotor; oriV - origin of replication for the bacterial F plasmid; ori2 - secondary origin of replication for the bacterial F plasmid (also known as oriS); repE - replication initiation protein for the bacterial F plasmid; incC - incompatibility region of the bacterial F plasmid; sopA - partitioning protein for the bacterial F plasmid; sopB - partitioning protein for the bacterial F plasmid; incP origin of transfer oriT - incP origin of transfer; ApramR - apramycin resistance gene; Factor Xa site - factor Xa recognition and cleavage site; phage phi-C31 attp - attachment site of phage phi-C31; phage phi-C31 integrase - integrase from phage phi-C31; sopC - sopB binding site. Primer sites for Gibson cloning of analogs as described in Examples 1 and 2 are indicated with designation of “Gibson.”

[0055] FIG. 6 shows a schematic illustration of ETBR mechanism for reducing anti- tumor immune response.

[0056] FIG. 7 shows exemplary binding curves for the parent lasso peptide (SEQ ID NO 1) vs human and mouse ETBR. Binding data is shown in Table 6.

[0057] FIG. 8 shows exemplary binding curves for an engineered lasso peptide having an F11Y substitution (SEQ ID NO 35) vs human and mouse ETBR. Binding data is shown in Table 6 [0058] FIG. 9 shows exemplary binding curves for an engineered lasso peptide having an F12H substitution (SEQ ID NO 39) vs human and mouse ETBR. Binding data is shown in Table 6

[0059] FIG. 10 shows exemplary binding curves for an engineered lasso peptide having an F12W substitution (SEQ ID NO 42) vs human and mouse ETBR. Binding data is shown in Table 6.

[0060] FIG. 11 shows exemplary binding curves for an engineered lasso peptide having an F12Y substitution (SEQ ID NO 43) vs human and mouse ETBR. Binding data is shown in Table 6.

[0061] FIG. 12 shows exemplary binding curves for an engineered lasso peptide having an H4M substitution (SEQ ID NO 13) vs human and mouse ETBR. Binding data is shown in Table 6

[0062] FIG. 13 shows exemplary binding curves for an engineered lasso peptide having F11Y and F12Y substitutions (SEQ ID NO 61) vs human and mouse ETBR. Binding data is shown in Table 6.

7. DETAILED DESCRIPTION

[0063] The novel features of the subject matter described herein are set forth specifically in the appended claims. A better understanding of the features and benefits of the present disclose will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the claimed embodiments are utilized. To facilitate a full understanding of the disclosure set forth herein, a number of terms are defined below.

7.1 General Techniques

[0064] Techniques and procedures described or referenced herein include those that are generally well understood and/or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al.. Molecular Cloning: A Laboratory Manual (4th ed. 2012); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); Antibody Engineering Vols 1 and 2 (Kontermann and Diibel eds., 2nd ed. 2010); Molecular Biology of the Cell (6th Ed., 2014); Organic Chemistry, (Thomas Sorrell, 1999); March's Advanced Organic Chemistry (6^th ed. 2007); Lasso Peptides, (Li, Y.; Zirah, S.; Rebuffet, S., Springer; New York, 2015); Natural Products in Medicinal Chemistry, Methods and Principles in Medicinal Chemistry (Hanessian, S., ed., Wiley-VCH; 1st edition, 2014); and Basic Principles of Drug Discovery and Development (Blass, B. Academic Press; 1st edition, 2015).

7.2 Terminology

[0065] Unless described otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of term set forth below shall control.

Conventions and Abbreviations

[0066] The singular terms “a,” “an,” and “the” as used herein include the plural reference unless the context clearly indicates otherwise. [0067] The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0068] The terms “administer” and “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an engineered lasso peptide as described herein) into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art. When a disease, disorder, condition, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease, disorder, condition, or symptoms thereof. When a disease, disorder, condition, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease, disorder, condition, or symptoms thereof. When two or more substances are being administered, this can be referred as co-administration and refers to the simultaneous or sequential administration of at least two substances according to the present disclosure. For example, an engineered lasso peptide as disclosed herein can be administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. When substances are administered repeatedly to maintain the initial therapeutic effect (activity) in a continuous mode (e.g., for a period of time such as days, weeks, months, or years), such administration can be referred to as chronic administration, which is contrast to an acute mode. Another type of administration includes intermittent administration, which is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

[0069] The term “aerobic” when used in reference to a culture or growth condition means that free oxygen (O2) is available in the culture or growth condition. This includes when the dissolved oxygen in the liquid medium is more than 50% of saturation.

[0070] The term “alteration” or grammatical equivalents thereof as used herein when used in reference to any peptide, polypeptide, protein, nucleic acid or polynucleotide described herein refers to a change in structure of an amino acid residue, an amino acid sequence, a nucleic acid base, or an nucleic acid sequence relative to the starting or reference residue, base or sequence. An alteration of an amino acid residue includes, for example, deletions, insertions and substitutions. The term “substitution” when used in reference to a peptide, polypeptide, protein refers to an amino acid residue that has been substituted for a structurally different amino acid residue. Such substitutions can be a conservative substitution, a non-conservative substitution, a substitution to a specific sub-class of amino acids, or a combination thereof as described herein. An alteration of a nucleic acid base includes, for example, changing one naturally occurring base for a different naturally occurring base, such as changing an adenine to a thymine or a guanine to a cytosine or an adenine to a cytosine or a guanine to a thymine. An alteration of a nucleic acid base can result in an alteration of the encoding peptide, polypeptide or protein by changing the encoded amino acid residue or function of the peptide, polypeptide or protein. An alteration of a nucleic acid base may not result in an alteration of the amino acid sequence or function of encoded peptide, polypeptide or protein, also known as a silent mutation.

[0071] The term “amino acid” refers to naturally occurring and non-naturally occurring alpha-amino acids, as well as alpha-amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring alpha-amino acids. Naturally encoded amino acids are the 22 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, pyrrolysine and selenocysteine). Amino acid analogs or derivatives refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and a side chain R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms “non- natural amino acid” or “non-proteinogenic amino acid” or “unnatural amino acid” or “non- canonical” refer to alpha-amino acids that contain different side chains (different R groups) relative to those that appear in the twenty -two common or naturally occurring amino acids listed above. In addition, these terms also can refer to amino acids that are described as having D-stereochemistry, rather than L-stereochemistry of natural amino acids, despite the fact that some amino acids do occur in the D-stereochemical form in nature (e.g., D-alanine and D-serine).

[0072] The terms “antagonize” and “antagonist” when used in reference to an engineered lasso peptide refer to one which inhibits or reduces biological activity of the target molecule it binds or the pathway the target molecule mediates. For example, an engineered lasso peptide that specifically antagonizes the target molecule (e.g., ETBR) can substantially or completely inhibit the biological activity of the target molecule or the pathway the target molecule mediates.

[0073] The term “binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding protein such as an engineered lasso peptide) and its binding partner (e.g., a target protein). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., an engineered lasso peptide and target protein). The affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (K_D). Similarly, the affinity of an inhibiting molecule X for its binding partner Y can generally be represented by the inhibition constant (Ki). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity lasso peptides generally bind target proteins slowly and tend to dissociate readily, whereas high-affinity lasso peptides generally bind target proteins faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure.

Specific illustrative embodiments include the following: the “K_D” or “K_D value” can be measured by assays known in the art, for example by a binding assay, including in a radioimmunoassay (RIA), a surface plasmon resonance assay as provided by Biacore®, using, for example, a Biacore ®TM-2000 or a Biacore ®TM-3000, or by biolayer interferometry using, for example, the Octet®QK384 system, performed with the engineered lasso peptide described herein and its target protein (e.g., ETBR); an “on-rate” or “rate of association” or “association rate” or “k_On” can also be determined with the same surface plasmon resonance or biolayer interferometry techniques described above using, for example, a Biacore ®TM- 2000 or a Biacore®TM-3000, or the Octet®QK384 system; and the “Ki” or “Ki value” can be measured by assays known in the art, for example, direct estimation of Ki and rate of enzyme inactivation (kinact) from time-dependent IC₅₀ values as described in Krippendorff et al., J. Biomolecular Screening, 14(8): 913-923 (2009) or an endpoint competition assay as described in Miyahisa et al., Angew Chem Int Ed Engl. 54(47): 14099-14102 (2015). [0074] The terms “binds” and “binding” refer to an interaction between molecules (e.g., nucleic acids, oligonucleotides, proteins, polypeptides, or peptides) including, for example, the formation of a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non- covalent interactions between a single target-binding site of a binding molecule (e.g., protein, polypeptide, or peptide) and a single target site of a target molecule is the affinity of the binding molecule for that target site. For example, the ratio of dissociation rate (koff) to association rate (k_on) of a binding protein (e.g., an engineered lasso peptide) to a monovalent target site (k_Off/k_On) is the dissociation constant K_D, which is inversely related to affinity. The lower the K_D value, the higher the affinity of the binding protein. The value of K_D varies for different complexes of binding molecules and target molecules and depends on both k_on and koff. When the binding of a binding protein results in inhibition of the target molecule, such binding can be described by an inhibition constant (Ki). The Ki also represents a K_D, but more narrowly for the binding of an inhibitor to a target molecule; a binding protein whose binding reduces the activity of the target molecule. The binding equilibrium described by the Ki value depends on the kinetic mechanism of inhibition. The dissociation constant K_D or inhibition constant Ki for a binding protein (e.g., an engineered lasso peptide) provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. In general, the Ki value is used whenever the binding constant is measured through inhibition kinetics, while the K_D value is preferred when the binding is measured more directly (e.g., by fluorescence quenching, isothermal titration calorimetry, or surface plasmon resonance). The affinity at one binding site does not always reflect the true strength of the interaction between a binding protein and the target molecule. When complex target molecule containing multiple, repeating target sites, such as a polyvalent target protein, come in contact with lasso peptides containing multiple target binding sites, the interaction of the lasso peptide with the target protein at one site will increase the probability of a reaction at a second site.

[0075] The term “carriers” as used herein includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; 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™, polyethylene glycol (PEG), and PLURONICS™. The term “carrier” can also refer to a diluent, adjuvant (e.g., Freund’s adjuvant (complete or incomplete)), excipient, or vehicle. Such carriers, including pharmaceutical carriers, can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary carrier when a composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral compositions, including formulations, can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington ’s Pharmaceutical Sciences (A.R. Gennaro, 19th ed. 1995, Mack Publishing Company). Compositions, including pharmaceutical compounds, can contain an engineered lasso peptide, for example, in isolated or purified form, together with a suitable amount of carriers.

[0076] The term “chemotherapy” as used herein refers to systemic treatment a subject suffering from or at risk of suffering from cancer with one or more anticancer drugs. Types of chemotherapy include adjuvant chemotherapy (treatment of a patient after the primary tumor has been removed and there is no evidence that cancer remains in the body; given to improve survival), primary chemotherapy (also referred to as neoadjuvant chemotherapy; treatment of a cancer with an anticancer drug as the primary treatment or prior to surgery or radiation), or combination chemotherapy (the use of two or more anticancer drugs to treat a patient). Anticancer drugs that can be used for chemotherapy include alkylating agents (e.g., cyclophosphamide and mustargen), platinum drugs (e.g., cisplatin, carboplatin, and oxaliplatin), antimetabolites (e.g., 5-fluorouracil, tegafur, and uracil), antibiotics (e.g., doxorubicin, daunorubicin, idarubicin, epirubicin, dactinomycin, and bleomycin), topoisomerase inhibitors (e.g., etoposide, teniposide, topotecan, and irinotecan), antimicrotubule agents (e.g., vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, and estramustine phosphate) and hormones (e.g., tamoxifen, leuprolide acetate, and goserelin). [0077] The term “compete” when used in the context of lasso peptides (e.g., an engineered lasso peptide and other binding proteins that bind to and compete for the same target molecule or target site on the target molecule) means competition as determined by an assay in which the engineered lasso peptide under study prevents or inhibits the specific binding of a reference molecule (e.g., a reference ligand of the target molecule) to a common target molecule. Numerous types of competitive binding assays can be used to determine if a test lasso peptide competes with a reference ligand for binding to a target molecule.

Examples of assays that can be employed include solid phase direct or indirect RIA, solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-53), solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, . Immunol. 137:3614-19), solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, Antibodies, A Laboratory Manual (1988)), solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., 1988, Mol. Immunol. 25:7-15), and direct labeled RIA (see, e.g., Moldenhauer et al., 1990, Scand. I. Immunol. 32:77-82). Typically, such an assay involves the use of a purified target molecule bound to a solid surface, or cells bearing either of an unlabeled test target- binding lasso peptide or a labeled reference target-binding protein (e.g., reference target- binding ligand). Competitive inhibition can be measured by determining the amount of label bound to the solid surface in the presence of the test target-binding lasso peptide. Usually the test target-binding protein is present in excess. Target-binding lasso peptides identified by competition assay (e.g., competing lasso peptides) include lasso peptides binding to the same target site as the reference and lasso peptides binding to an adjacent target site sufficiently proximal to the target site bound by the reference for steric hindrance to occur. Additional details regarding methods for determining competitive binding are described herein. Usually, when a competing lasso peptide is present in excess, it will inhibit specific binding of a reference to a common target molecule by at least 30%, for example 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more.

[0078] The term “condition suitable for lasso formation,” depending on the context, can refer to, for example, a condition suitable for the expression of one or more protein products in a bacterial host (e.g., a lasso precursor peptide, or a processing enzyme). Exemplary suitable conditions include, but are not limited to, a suitable culturing condition of the bacterial host that enable the protein synthesis and transportation in the host cell. Additionally or alternatively, depending on the context, the term “condition suitable for lasso formation” can refer to, for example, a condition suitable for post-translational modification of a lasso precursor peptide. Exemplary suitable conditions include, but are not limited to, a suitable temperature and/or incubation time for a lasso cyclase and/or lasso peptidase to process the lasso precursor in to a matured lasso peptide.

[0079] The term “conjugate” as used herein refers to the joining together of two or more molecules by the formation of a covalent bond. The conjugation of two or more molecules can result in a heterologous molecule being formed (e.g., an engineered lasso peptide and a therapeutic agent).

[0080] The terms “downregulate” and “downregulation” as used herein refer to lowering the rate or level a molecule relative to a control. Downregulation of a molecule can be expressed as a percentage (e.g., 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99%) or by a fold change (i.e., 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 10 fold or more).

[0081] The term “EC₅₀” refers an amount, concentration, or dosage of a compound that results in for 50% of a maximal response in an assay that measures such response.

[0082] The term “effective amount” as used herein generally refers to an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate the symptoms and/or underlying cause, prevent the occurrence of symptoms and/or their underlying cause, and/or improve or remediate the damage that results from or is associated with a disease, disorder, or condition, including, for example, cancer. In some instances, the effective amount is a therapeutically effective amount or a prophylactically effective amount. [0083] The term “encoding nucleic acid” or grammatical equivalents thereof as it is used in reference to nucleic acid molecule refers to a nucleic acid molecule in its native state or when manipulated by methods well known to those skilled in the art that can be transcribed to produce mRNA, which is then translated into a polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid molecule, and the encoding sequence can be deduced therefrom.

[0084] The terms “engineered” and “variant” as used here in when used in reference to any peptide, polypeptide, protein, nucleic acid or polynucleotide described herein refer to a sequence of amino acids or nucleic acids having at least one alteration (e.g., substitution) at an amino acid residue or nucleic acid base as compared to a parent sequence. Such a sequence of amino acids or nucleic acids is not naturally occurring. The parent sequence of amino acids or nucleic acids can be, for example, a wild-type sequence or a homolog thereof, or a modified variant of a wild-type sequence or homolog thereof.

[0085] The term “excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.), and polyols (e.g., mannitol, sorbitol, etc.). See, also, Remington ’s Pharmaceutical Sciences (A.R. Gennaro, 19th ed. 1995, Mack Publishing Company), which is hereby incorporated by reference in its entirety.

[0086] The term “glucose-limiting” as used herein when used in reference to culturing conditions references to use of media having a minimal about of glucose needed for survival of the host cell. Such minimal glucose media can include media having no more than about 30 mM, no more than about 25 mM, no more than about 20 mM, no more than about 15 mM, no more than about 10 mM, no more than about 5 mM, no more than about 2 mM glucose.

[0087] The term “IC₅₀” refers an amount, concentration, or dosage of a compound that results in 50% inhibition of a maximal response in an assay that measures such response. [0088] The terms “inhibition” and “inhibit” when used herein refer to partial (such as, 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99%) or complete (i.e., 100%) inhibition.

[0089] The term “immunotherapy” as used herein refers to treatment of a subject suffering from or at risk of suffering from a disease by a method that includes inducing, enhancing, suppressing or other modification of an immune response by use of an immunomodulator. Immunomodulators that can be used for immunotherapy include interleukins, cytokines, chemokines, cytosine phosphate-guanosine, oligodeoxynucleotides and glucans, and cells, such as T cells, lymphocytes, macrophages, dendritic cells, natural killer cells, cytotoxic T lymphocytes, immune checkpoint modulators (e.g., immune checkpoint inhibitors or immune checkpoint stimulators), and vaccines (e.g., anti-cancer vaccines). For example, an immune checkpoint inhibitor works by blocking checkpoint proteins from binding with their partner proteins, thereby preventing the “off’ signal from being sent, allowing T cells to kill a target cell (e.g., a cancer cell). As another example, anti- cancer vaccines help a subject’s immune system to recognize and react to antigens that are specific to cancer cells (e.g., tumor-associated antigens). Use of such immunomodulators or cells supplements, enhances, replaces or otherwise modifies the subject's own inadequate or inappropriate immune response. In the context of cancer, immunotherapy can refer to stimulation of the immune system to reject and destroy tumors, for example, with cytokines or cells. Also in the context of cancer, adoptive immunotherapy involves the administration of cells having anti-tumor activity, including activated or expanded T cells, lymphocytes, macrophages, dendritic cells, natural killer cells and cytotoxic T lymphocytes. Such cells are administered to a subject with the aim that the cells mediate either directly or indirectly specific immunity to tumor cells and/or antigenic components or regression of the tumor. Active immunotherapy involves injection of cells or proteins to generate either new or enhance systemic immune responses to the administered cell or protein.

Passive immunotherapy involves the administration of an antibody.

[0090] The terms “isolated,” “isolate,” and “isolating,” or grammatical equivalent thereof, when used in reference to a microbial organism, nucleic acid, protein, polypeptide, or peptide, refer to a microbial organism, nucleic acid, protein, polypeptide, or peptide that is substantially free of at least one component relative to the referenced microbial organism, nucleic acid, protein, polypeptide, or peptide is found in nature or in its current environment. The term includes a microbial organism, nucleic acid, protein, polypeptide, or peptide that is removed from some or all components as it is found in its natural environment. Therefore, an isolated microbial organism, nucleic acid, protein, polypeptide, or peptide is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments (e.g., laboratories). Specific examples of isolated microbial organism, nucleic acid, protein, polypeptide, or peptide include a partially pure microbial organism, nucleic acid, protein, polypeptide, or peptide, a substantially pure microbial organism, nucleic acid, protein, polypeptide, or peptide, a microbial organism cultured in a medium that is non-naturally occurring, a protein, polypeptide, or peptide purified from other components and substances present their natural environment, including other proteins, polypeptides, or peptides, or an isolated nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. As another example, an isolated nucleic acid can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A substantially pure molecule can include isolated forms of the molecule.

[0091] The terms “lasso core peptide” and “core peptide” refer to the peptide or the peptide segment of the precursor peptide that is processed into or otherwise forms an engineered lasso peptide having the lariat-like topology. As used herein, a core peptide can have the same amino acid sequence as an engineered lasso peptide, but has not matured to have the lariat-like topology of an engineered lasso peptide. Core peptides can have different lengths of amino acid sequences. For example, the core peptide of the engineered lasso peptide described herein is 16 amino acids long, but other core peptides can be, for example, about 10 amino acids long to as many as about 65 amino acids long.

[0092] The term “lasso cyclase” as used herein refers to the enzyme capable of catalyzing cyclization of the ring portion of a lasso core peptide.

[0093] The term “lasso peptidase” as used herein refers to the enzyme capable of catalyzing the removal of the leader sequence from a lasso precursor peptide to produce a lasso peptide (e.g., an engineered lasso peptide).

[0094] The terms “lasso peptide” and “lasso” are used interchangeably herein, and is used to refer to a class of peptide or polypeptide having the general lariat-like topology as exemplified in FIG. 1. As shown in the figure, the lariat-like topology can be generally divided into a ring portion, a loop portion, and a tail portion. Particularly, a region on one end of the peptide forms the ring around the tail on the other end of the peptide, the tail is threaded through the ring, and a middle loop portion connects the ring and the tail, together forming the lariat-like topology. Particularly, the amino acid residues that are joined together to form the ring are herein referred to as the “ring-forming amino acid.” A ring-forming amino acid can located at the N- or C- terminus of the lasso peptide (“terminal ring-forming amino acid”), or in the middle (but not necessarily the center) of a lasso peptide (“internal ring-forming amino acid”). An engineered lasso peptide can be referred to here as being “cyclized” when such a lariat-like topology is formed by the engineered lasso peptide. For example, the term “G1-D9 cyclized” as used herein when referring to a lasso peptide, means that the lasso peptide has a N-terminal ring -forming amino acid of a glycine residue (Gl) and an internal ring-forming amino acid of an aspartate residue at position 9 (D9), where the amino group of Gl and the carboxyl group of D9 form an isopeptide bond, thus forming the ring portion of the lasso peptide. The fragment of a lasso peptide between and including the two ring-forming amino acid residues is the ring portion. The fragment of a lasso peptide between the internal ring-forming amino acid and where the peptide threaded through the plane of the ring is the loop portion. The remaining fragment of a lasso peptide starting from where the peptide is threaded through the plane of the ring is the tail portion. In addition to the lariat-like topology, additional topological features of a lasso peptide can further include intra-peptide disulfide bonding, such as disulfide bond(s) between the tail and the ring, between the ring and the loop, and/or between different locations within the loop or tail. As used herein, “lasso peptide” or “lasso” refers to both naturally-existing peptides and artificially produced peptides that have the lariat-like topology as described herein. Similarly, an “engineered lasso peptide” or “engineered lasso” refers to non-naturally occurring analogs, derivatives, or variants of a naturally occurring lasso peptide, which analogs, derivatives or variants are also lasso peptides themselves.

[0095] The term “lasso peptide biosynthesis component” as used herein refers to a protein comprising one or more of (i) a lasso peptidase, (ii) a lasso cyclase, and (iii) RRE. An exemplary process of lasso peptide production using lasso peptide biosynthesis components from a lasso precursor peptide is depicted in FIG. 3 and FIG. 4.

[0096] The terms “lasso precursor peptide” or “precursor peptide” as used herein refer to a peptide that is processed into or otherwise forms a lasso peptide. A lasso precursor peptide can include at least one engineered lasso core peptide portion. A lasso precursor peptide can also include one or more amino acid residues or amino acid fragments that do not belong to an engineered lasso core peptide, such as a leader sequence that facilitates recognition of the lasso precursor peptide by one or more lasso processing enzymes.

[0097] The term “leader sequence” as used in reference to a lasso peptide, such as a lasso precursor peptide, refers to an amino acid sequence that facilitates recognition and processing by the lasso peptide processing enzymes described herein to form a cyclized lasso peptide. Accordingly, a lasso core peptide (e.g., an engineered lasso peptide) having a leader sequence can be referred to as a lasso precursor peptide.

[0098] The terms “manage,” “managing,” and “management” refer to the beneficial effects that a subject derives from a therapy (e.g., a prophylactic or therapeutic agent), which does not result in a cure of the disease. In some instances, a subject is administered one or more therapies (e.g., prophylactic or therapeutic agents, such as an engineered lasso peptide provided herein) to “manage” an endothelin B receptor-mediated proliferative disease (e.g., cancer), one or more symptoms thereof, so as to prevent the progression or worsening of the disease.

[0099] The term “maximal percent downregulation” as used herein refers to the maximal level of downregulation achievable based a dose-response curve in an assay that measures such a response.

[00100] The term “maximal percent inhibition” as used herein refers to the maximal level of inhibition achievable based a dose-response curve in an assay that measures such a response.

[00101] The term “maximal percent reduction” as used herein refers to the maximal level of reduction achievable based a dose-response curve in an assay that measures such a response.

[00102] The terms “microbial,” “microbial organism” and “microorganism” as used herein refer to any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cells of any species that can be cultured for the production of a biochemical (e.g., a lasso peptide, including an engineered lasso peptide).

[00103] The term “microenvironment” of a neoplastic cell or neoplastic cells or “neoplastic microenvironment” refers to elements of the neoplasia milieu that creates a structural and/or functional environment for the neoplastic process to survive, expand, and/or spread. As a non-limiting example, a neoplastic microenvironment is constituted by the cells, molecules, extracellular matrix and/or blood vessels that surround and/or feed one or more neoplastic cells, such as a solid tumor. In certain embodiments, the neoplastic disease is a solid tumor. Exemplary cells or tissue within the tumor microenvironment include, but are not limited to, tumor vasculature, tumor infiltrating lymphocytes, fibroblast reticular cells, endothelial progenitor cells (EPC), cancer-associated fibroblasts, pericytes, other stromal cells, components of the extracellular matrix (ECM), dendritic cells, antigen presenting cells, T-cells, regulatory T-cells, macrophages, neutrophils, and other immune cells located proximal to a tumor. Exemplary cellular functions affecting the tumor microenvironment include, but are not limited to, production of cytokines and/or chemokines, response to cytokines, antigen processing and presentation of peptide antigen, regulation of leukocyte chemotaxis and migration, regulation of gene expression, complement activation, regulation of signaling pathways, cell-mediated cytotoxicity, cell-mediated immunity, humoral immune responses, and innate immune responses, etc.

[00104] The terms “modulating” and “modulate” as used herein refer to an effect of altering a biological activity (i.e. increasing or decreasing the activity), especially a biological activity associated with a particular biomolecule such as a, enzyme or cell surface receptor. For example, an inhibitor of a particular biomolecule modulates the activity of that biomolecule, e.g., an enzyme, by decreasing the activity of the biomolecule, such as an enzyme. Such activity is typically indicated in terms of an inhibitory concentration (IC₅₀) of the compound for an inhibitor with respect to, for example, an enzyme or a cell surface receptor.

[00105] The terms “naturally occurring,” “natural,” and “native” when used in connection with naturally occurring biological materials, such as nucleic acid molecules, oligonucleotides, amino acids, polypeptides, peptides, metabolites, small molecule natural products, host cells, and the like, refer to materials that are found in or isolated directly from Nature and are not changed or manipulated by humans.

[00106] The terms “non-naturally occurring,” “non-natural,” “unnatural” and “non-native” as used herein refer to a material, substance, molecule, cell, nucleic acid, oligonucleotide, nucleotide, enzyme, protein, polypeptide, peptide, or amino acid that is not known to exist or is not found in Nature or that has been structurally modified and/or synthesized by humans. Such terms when used in reference to a microbial organism, cell extract, or nucleic acid of the disclosure mean that the microbial organism, cell extract, or nucleic acid has at least one genetic alteration not normally found in a naturally occurring strain or a naturally occurring nucleic acid of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, introduction of expressible oligonucleotides or nucleic acids encoding polypeptides (e.g., an engineered lasso peptide), nucleic acid additions, substitutions, or deletions and/or other functional disruption of the microbial organism’s genetic material. Such alterations include, for example, nucleotide changes, additions, substitutions or deletions in the genomic coding regions and functional fragments thereof, used for heterologous, homologous or both heterologous and homologous expression of polypeptides. Additional alterations include, for example, nucleotide changes, additions, substitutions or deletions in the genomic non-coding and/or regulatory regions in which the modifications alter expression of a gene or operon. Such terms when used in reference to a protein, polypeptide, or peptide are used to refer to a protein, polypeptide, or peptide having amino acids that are introduced into the amino acid sequence of the protein, polypeptide, or peptide to modify the properties of the polypeptide.

[00107] The terms “oligonucleotide” and “nucleic acid” refer to oligomers of deoxyribonucleotides (e.g., DNA) or ribonucleotides (e.g., RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also refers to oligonucleotide analogs including PNA (peptidonucleic acid), analogs of DNA used in antisense technology (phosphorothioates, phosphoroamidates, and the like). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer, M.A., et al., Nucleic Acid Res., 1991, 19, 5081-1585;

Ohtsuka, E. et al., J. Biol. Chem., 1985, 260, 2605-2608; and Rossolini, G.M., et al., Mol. Cell. Probes, 1994, 8, 91-98). “Oligonucleotide,” as used herein, refers to short, generally single-stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides. A cell that produces an engineered lasso peptide of the present disclosure can include a bacterial and archaea host cells into which nucleic acids encoding the lasso peptide component have been introduced. Suitable host cells are disclosed below.

[00108] Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double- stranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3 ’ to the 3 ’ end of the RNA transcript are referred to as “downstream sequences.”

[00109] The term “operatively linked” as used herein when used in reference to a nucleic acid encoding a protein, polypeptide, or peptide refers to connection of a nucleotide sequence encoding the protein, polypeptide, or peptide to another nucleotide sequence (e.g., a promoter) is such a way as to allow for the connected nucleotide sequences to function (e.g., express the protein, polypeptide, or peptide in the host).

[00110] The term “peptide” as used herein refers to a polymer chain containing between two and fifty (2-50) amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non- naturally occurring amino acid, e.g., an amino acid analog or non-natural amino acid.

[00111] The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

[00112] The terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of greater than about fifty (50) amino acid residues. That is, a description directed to a polypeptide applies equally to a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid, e.g., an amino acid analog. As used herein, the terms encompass amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.

[0100] The term “post-translationally modified peptide (RiPP) recognition element” or “RRE” refers to a facilitator protein element encoded in some lasso gene clusters that facilitates the recognition of and binding to the leader sequence by the lasso peptidase during biosynthesis of the lasso peptide.

[00113] The terms “preferential binding” and “preferentially binds to” when used in reference to a particular polypeptide or peptide (an engineered lasso peptide) on a particular target molecule (e.g., ETBR) with respect to a reference molecule (e.g., ETAR) refers to binding of the target molecule that is measurably higher than binding of the reference molecule, while the reference molecule may or may not also bind to the engineered lasso peptide. For example, an engineered lasso peptide described herein can preferentially binds to ETBR over ETAR. Preferential binding can be determined, for example, by determining the binding affinity for the target molecule and the reference molecule. For example, an engineered lasso peptide that preferentially binds to a target molecule over a reference molecule can bind to the target molecule with a K_D less than the K_D exhibited relative to the reference molecule.

[00114] An engineered lasso peptide that specifically or preferentially binds to a target protein (e.g., ETBR) can be identified, for example, by immunoassays (e.g., ELISA, fluorescent immunosorbent assay, chemiluminescence immune assay, radioimmunoassay (RIA), enzyme multiplied immunoassay, solid phase radioimmunoassay (SPRIA), a surface plasmon resonance (SPR) assay (e.g., Biacore®), a fluorescence polarization assay, a fluorescence resonance energy transfer (FRET) assay, Dot-blot assay, fluorescence activated cell sorting (FACS) assay, or other techniques known to those of skill in the art. Typically, a specific or selective reaction will be at least twice background signal or noise and can be more than 10 times background.

[00115] The terms “prevent,” “preventing,” and “prevention” refer to delaying or reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s) (e.g., cancer).

[00116] The terms “proliferative” and “neoplastic” disease or condition refer to any condition in animals that is characterized by uncontrolled, abnormal growth of cells, referred to as “neoplastic cells.” Neoplastic cells, as used herein, can be malignant or benign, and includes both solid tumors as well as hematologic tumors and/or malignancies. Non-limiting examples of proliferative diseases that can be prevented, treated or managed with the methods and compositions described herein include those mediated by endothelin B receptor activity, such as cancer, including breast cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), hepatocellular carcinoma, prostate cancer, ovarian cancer, gastric cancer, brain or spinal cancer (e.g., glioma, such as a glioblastoma), melanoma, cancer of the head and neck, colorectal cancer, bladder cancer, vulvar cancer, esophageal squamous cell carcinoma, renal cancer (e.g., clear-cell renal cell carcinoma), cervical cancer, salivary gland carcinoma, lung cancer (e.g., non-small cell lung cancer and small-cell lung cancer), multiple myeloma, or Kaposi’s sarcoma. A proliferative or neoplastic disease or condition when cancer includes a primary cancer as well as a metastatic cancer that is derived from a primary cancer tumor.

[00117] The term “promoter” as used herein in reference to a nucleic acid encoding a protein, polypeptide or peptide refers to a nucleotide sequence where transcription of a linked open reading frame (e.g., a nucleotide sequence encoding an engineered lasso peptide) by an RNA polymerase begins. A promoter sequence can be located directly upstream or at the 5' end of the transcription initiation site. RNA polymerase and the necessary transcription factors bind to a promoter sequence and initiate transcription. Promoter sequences define the direction of transcription and indicate which DNA strand will be transcribed, i.e. the sense strand.

[00118] The term “prophylactic agent” refers to any agent that can totally or partially inhibit the development, recurrence, onset, or spread of an endothelin B receptor-mediated proliferative disease (e.g., cancer) and/or symptom related thereto in a subject. In some istances, the term “prophylactic agent” refers to an engineered lasso peptide as described herein.

[00119] The term “prophylactically effective amount” refers to an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing, delaying, or reducing the likelihood of the onset (or reoccurrence) of a disease, disorder, condition, or associated symptom(s) (e.g., cancer). Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of a disease, disorder, or condition, a prophylactically effective amount can be less than a therapeutically effective amount. The full therapeutic or prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more administrations.

[00120] The term “recombinant” as used herein with respect to a nucleic acid, such as a nucleic acid comprising a gene that encodes a protein or polypeptide (e.g., an engineered lasso peptide described herein), refers to: a nucleic acid that has been artificially supplied to a biological system; a nucleic acid that has been modified within a biological system, or a nucleic acid whose expression or regulation has been manipulated within a biological system. The recombinant nucleic acid can be supplied to the biological system, for example, by introduction of the nucleic acid into genetic material of a microbial organism, such as by integration into a microbial organism chromosome, or as non-chromosomal genetic material such as a plasmid. A recombinant nucleic acid that is introduced into or expressed in a microbial organism can be a nucleic acid that comes from a different organism or species from the microbial organism, or can be a synthetic nucleic acid, or can be a nucleic acid that is also endogenously expressed in the same organism or species as the microbial organism. A recombinant nucleic acid that is also endogenously expressed in the same organism or species as the microbial organism can be considered heterologous if: the sequence of the recombinant nucleic acid is modified relative to the endogenously expressed sequence, the sequence of a regulatory region such as a promoter that controls expression of the nucleic acid is modified relative to the regulatory region of the endogenously expressed sequence, the nucleic acid is expressed in an alternate location in the genome of the microbial organism relative to the endogenously expressed sequence, the nucleic acid is expressed in a different copy number in the microbial organism relative to the endogenously expressed sequence, and/or the nucleic acid is expressed as non-chromosomal genetic material such as a plasmid in the microbial organism.

[00121] The terms “selective inhibition of’ and “selectively inhibits” as used herein with regard to inhibition of a target molecule by an engineered lasso peptide refer to inhibition of the target molecule activity is measurably stronger than inhibition of a reference molecule activity. For example, in some instances, an engineered lasso peptide selectively inhibits ETBR over ETAR. Selective inhibition can be determined, for example, by determining the IC₅₀ value. For example, an engineered lasso peptide that selectively inhibits a target molecule can exhibit an IC₅₀ value less than the IC₅₀ exhibited relative to a reference molecule. In some instances, the engineered lasso peptide selectively inhibits a target molecule with an IC₅₀ less than half of the IC₅₀ exhibited relative to the reference molecule. In some instances, the lasso peptide selectively inhibits a target molecule with an IC₅₀ that is about 75%, about 50%, about 25%, about 10%, about 5%, about 2.5%, or about 1% of the IC₅₀ exhibited relative to the reference molecule. In some instances, the ratio between the IC₅₀ exhibited by the lasso peptide with respect to the reference molecule and the IC₅₀ exhibited with respect to the target molecule is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 100 fold, at least 500 fold, at least 10³ fold, at least 10⁴ fold, or at least 10⁵ fold.

[00122] The terms “soluble” and “solubility” as used herein in reference to a protein, polypeptide or peptide refer to the concentration of aqueous soluble protein in equilibrium with the solid phase of the protein under a given condition (e.g., pH, temperature, and solvent composition). The surface of a protein, polypeptide or peptide has a net charge that depends on the number and identities of the charged amino acids, and on pH. At a specific pH the positive and negative charges will balance and the net charge will be zero. This pH is called the isoelectric point, and for most proteins, polypeptides and peptides it occurs in the pH range of 5.5 to 8. A protein, polypeptide or peptide has its lowest solubility at its isoelectric point. If there is a charge at the surface, the protein, polypeptide or peptide prefers to interact with water, thereby making it more soluble. Without a net charge, precipitation (the solid phase of the protein) is more likely.

[00123] With regard to the binding of an engineered lasso peptide to a target molecule, the terms “specific binding,” “specifically binds to,” and “is specific for” when used in reference to a particular molecule (e.g., protein, polypeptide, or peptide) refer to binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The terms also include binding where a molecule (e.g., protein, polypeptide, or peptide) binds to a particular protein or fragment of a particular protein without substantially binding to any other protein or protein fragment. Accordingly, an engineered lasso peptide described herein can be described as specifically binding to a target protein when it binds to the target protein with higher affinity than to any cross-reactive target protein, as determined using experimental techniques described herein.

[00124] The terms “stable” and “stability” as used herein in reference to a protein, polypeptide or peptide refer the quality, state, or degree of the protein, polypeptide or peptide as not changing. In particular, such quality, state, or degree of the protein, polypeptide or peptide can be in terms of degradation of the protein, polypeptide or peptide, which can be measured using well know methods in the art, including being measured by thermal degradation or proteolytic degradation through hydrolysis of a peptide bond.

[00125] The terms “subject” and “patient” are used interchangeably. As used herein, in some instances, a subject is a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, mouse, etc.) or a primate (e.g., monkey and human). In some instances, the subject is a human. In some instances, the subject is a mammal (e.g., a human or mouse) having an endothelin B receptor (ETBR)-mediated proliferative disease e.g. cancer), disorder, or condition. In some instances, the subject is a mammal (e.g., a human or mouse) at risk of developing an endothelin B Receptor (ETBR)-mediated proliferative disease e.g. cancer), disorder, or condition.

[00126] The term “substantially” means that something takes place, as a function or activity, to provide the expected outcome or result to a large degree and to a great extent, but still not to the fullest extent. For example, if an engineered lasso peptide is substantially purified, the engineered lasso peptide is isolated and purification steps afford the engineered lasso peptide at purity level above 80%, above 90%, above 95%, or above 98%, and as high as 99.99%.

[00127] The term “substantially all” refers to at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[00128] The term “therapeutic agent” refers to any agent that can be used in treating, preventing, or alleviating a disease, disorder, or condition, including in the treatment, prevention, or alleviation of one or more symptoms of an endothelin B receptor-mediated proliferative disease (e.g., cancer), disorder, or condition and/or a symptom related thereto. In some instances, a therapeutic agent refers to an engineered lasso peptide as described herein.

[00129] The term “therapeutically effective amount” as used herein refers to the amount of an agent (e.g., an engineered lasso peptide provided herein or any other agent described herein) that is sufficient to reduce and/or ameliorate the severity and/or duration of a given disease, disorder, or condition, and/or a symptom related thereto (e.g., cancer). A therapeutically effective amount of a substance/molecule/agent of the present disclosure (e.g., an engineered lasso peptide) can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule/agent to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance/molecule/agent are outweighed by the therapeutically beneficial effects. A therapeutically effective amount also encompasses an amount of an engineered lasso peptide or other agent (e.g., drug) effective to “treat” a disease, disorder, or condition, in a subject or mammal. [00130] The term “therapy” refers to any protocol, method, and/or agent that can be used in the prevention, management, treatment, and/or amelioration of an endothelin B receptor- mediated proliferative disease (e.g., cancer), disorder, or condition. In some instances, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and/or other therapies useful in the prevention, management, treatment, and/or amelioration of an endothelin B receptor-mediated proliferative disease (e.g., cancer), disorder, or condition, known to one of skill in the art such as medical personnel.

[00131] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some instances, treatment can be administered after one or more symptoms have developed. In other instances, treatment can be administered in the absence of symptoms. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment can also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

[00132] The terms “upregulate” and “upregulation” as used herein refer to increasing the rate or level a molecule relative to a control. Upregulation of a molecule can be expressed as a percentage (e.g., 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99%) or by a fold change (i.e., 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 10 fold or more).

[00133] The term “vasculature” refers to blood and lymph vessels that carry whole blood and lymphatic fluids. Accordingly, tumor vasculature refers to blood or lymph vessels that feed into a tumor.

[00134] The term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including, for example, a nucleic acid sequence encoding an engineered lasso peptide, a lasso precursor peptide (e.g., an engineered lasso peptide comprising a leader sequence), or lasso processing enzymes as described herein, in order to introduce the nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., both an engineered lasso core peptide and a lasso cyclase), both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product (e.g., an engineered lasso peptide as described herein), and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[00135] The term “wild-type” refers to organisms, cells, genes, biosynthetic gene clusters, enzymes, proteins, oligonucleotides, and the like that are found in Nature and are unchanged relative to these components found in Nature (in the wild).

7.3 Endothelin Receptors

[00136] In certain aspects of the present disclosure, the engineered lasso peptides provided herein are endothelin B receptor (ETBR) antagonistic compounds and their uses include managing, preventing, and/or treating an endothelin B receptor (ETBR)-mediated proliferative disease in a subject.

[00137] Endothelin (ET) receptors are transmembrane G protein-coupled receptors (GPCRs) normally expressed on the surface of endothelial cells lining the inner wall of blood and lymphatic vessels. Two main receptors, endothelin receptor type A (ETAR) and endothelin receptor type B (ETBR), regulate normal vascular function by binding to one of three cognate endothelin ligands, comprised of the 21-amino acid peptides endothelin-1 (ET- 1), endothelin-2 (ET-2), or endothelin-3 (ET-3). The vascular endothelium is an abundant source of the components of the endothelin axis; however, they also are expressed to varying extents by leukocytes, smooth muscle cells, mesangial cells, cardiac myocytes, and astrocytes. In humans, ETAR is located in the vasculature and is mostly expressed by cells of the vascular smooth muscle lineage. In these cells, binding of ET-1 to ETAR mainly induces vasoconstriction and cell proliferation (Maguire, J. J., Davenport, A.P., Endothelium receptors and their antagonists, Sem. Nephrology, 2015, 35(2), 125-136).

[00138] The terms “endothelin receptor type B” and “ETBR,” as used herein, refers to the endothelin receptor type B from any vertebrate source, including mammals such as primates (e.g., humans), dogs, and rodents (e.g., mice and rats), unless otherwise indicated. ETBR is a G protein-coupled receptor which activates a phosphatidylinositol-calcium second messenger system. The gene encoding human ETBR, referred to as EDNRB, is located on chromosome 13, at cytogenetic location 13q22.3. The amino acid sequence of human (homo sapiens) ETBR can be found at GenBank Accession No. NP 000106 and is provided herein as SEQ ID NO: 484. There are four identified mRNA transcript variants of human (homo sapiens) EDNRB that encode for human ETBR. An exemplary encoding nucleic acid sequence of human ETBR can be found at GenBank Accession No. NM 000115 and is provided herein as SEQ ID NO: 489. The amino acid sequence of mouse (Mus musculus) ETBR can be found at GenBank Accession No. NP_031930.1 and is provided herein as SEQ ID NO: 485. There are three identified mRNA transcript variants of mouse (Mus musculus) EDNRB that encode for ETBR. An exemplary encoding nucleic acid sequence of mouse ETBR can be found at GenBank Accession No. NM_007904.4 and is provided herein as SEQ ID NO: 490. [00139] ETBR in the vasculature is mostly expressed by endothelial cells. In these cells, binding of ETs to ETBR induces vasodilatation, bronchoconstriction, and cell proliferation. The human kidney is unusual among the peripheral organs in expressing a high density of ETBR. The renal vascular endothelium only expresses the ETBR subtype and ET-1 acts in an autocrine or paracrine manner to release vasodilators. Endothelial ETBR in kidney, as well as liver and lungs, appears to play a critical role in scavenging ET-1 from the plasma. The third major function for ET-1 is activation of ETBR in medullary epithelial cells to reduce salt and water reabsorption.

[00140] ET-1 can be induced in endothelial cells by many factors including mechanical stimulation, various hormones, and proinflammatory cytokines. Its production is inhibited by nitric oxide (NO), cyclic nucleotides, prostacyclin, and atrial natriuretic peptide (ANP). ET-1 also stimulates cardiac contraction and the growth of cardiac myocytes, regulates the release of vasoactive substances, and stimulates smooth muscle cell mitogenesis. ET-1 may control inflammatory responses by promoting the adhesion and migration of neutrophils and by stimulating the production of proinflammatory cytokines. [00141] ETAR and ETBR are GPCRs that transmit signals via heterotrimeric guanine nucleotide-binding G proteins, which are composed of α-, β-, and γ-subunits on the inner membrane surface of the cells, and are key determinants of many signaling processes, including signaling that leads to cell proliferation, apoptosis, survival, contraction, migration, and/or differentiation (Cabrera-Vera, T. M., et al., Insights into G Protein Structure, Function, and Regulation. Endocr. Rev. 2003, 24, 765-781). GPCR ligands interact with many downstream effectors, including adenyl cyclases, phosphodiesterases, phospholipases, tyrosine kinases, and ion channels. The duration of the signal is modulated by the activity of the multiple GPCR-mediated signaling pathways, leading to diverse biological responses. At physiological concentrations, ET-1 and ET-2, but not ET-3, bind to ETAR receptors with comparable affinity (K_D(ET-I) = K_D(ET-2) ~ 20-60 pM, K_D(ET-3) ~ 6500 pM), whereas all three ET ligands bind ETBR receptors with similar affinity (K_D(ET-I) = K_D(ET-2) = K_D(ET- 3) ~ 15 pM).

[00142] Endothelin-induced intracellular signaling transduced by activated ETAR and ETBR, which together control vascular homeostasis by balancing vasoconstriction, vasodilation, angiogenesis, and lymphangiogenesis (Vignon-Zellweger, N., et al.. Endothelin and endothelin receptors in the renal and cardiovascular systems, Life Sciences, 2012, 91, 490-500). Consistent with these roles, extensive early work revealed the various roles of the ET system in cardiovascular and renal disorders (Tomobe et al... Effects of endothelin on the renal artery from spontaneously hypertensive and Wistar Kyoto rats, Eur. J Pharmacol., 1988, 152(3): 373-374; Lehrke et al., Renal endothelin-1 and endothelin receptor type B expression in glomerular diseases with proteinuria. J Am Soc Nephrol., 2001, 12(11): 2321- 2329, Feldstein et al., Role of endothelins in hypertension. Am J Ther., 2007, 14(2): 147-153; Iglarz M et al., Mechanisms of ET-1 -induced endothelial dysfunction. J Cardiovasc Pharmacol., 2007, 50(6): 621-628) and the ET axis was targeted by therapeutic intervention largely for these diseases.

[00143] ETBR activation specifically mediates the release of relaxing factors such as nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor, increases in [Ca²⁺]i, protein kinase C, mitogen-activated protein kinase, and other pathways involved in vascular contraction and cell growth (Mazzuca, M.Q., Khalil, R.A. Vascular endothelin receptor type B: structure, function and dysregulation in vascular disease, Biochem Pharmacol. 2012; 84(2): 147-162). ETBR has been shown to be overexpressed in a variety of cancers and expression levels correlate with low survival and poor prognosis (Rosano, L., Bagnato, A., Endothelin therapeutics in cancer: Where are we? Am J Physiol Regul Integr Comp Physiol, 2016, 310: R469-R475; Bagnato, A., et al, Role of the endothelin axis and its antagonists in the treatment of cancer, Brit. J. Pharmacol., 2011, 163, 220-233). ETBR is overexpressed on the surface of tumor cells and promotes growth, proliferation, and metastasis of many tumor types, including esophageal squamous cell carcinoma (Ishimoto, S., et al., Role of endothelin receptor signalling in squamous cell carcinoma, Int. J. Oncology, 2012, 40, 1011- 1019; Tanaka, T., el al., Endothelin B receptor expression correlates with tumour angiogenesis and prognosis in oesophageal squamous cell carcinoma, Brit. J. Cancer, 2014, 110, 1027-1033), breast cancer (Grimshaw, M.J., et al., A Role for Endothelin-2 and Its Receptors in Breast Tumor Cell Invasion, Cancer Res., 2004, 64, 2461-2468; Wulfing, P., et al, Expression of endothelin-1, endothelin- A, and endothelin-B receptor in human breast cancer and correlation with long-term follow-up, Clin. Cancer Res., 2003, 9, 4125 4131), glioblastoma (Vasaiker, S., et al.. Overexpression of endothelin B receptor in glioblastoma: a prognostic marker and therapeutic target? BMC Cancer, 2018, 18: 154), oligodendroglioma (Wan, X., et al., Role of endothelin B receptor in oligodendroglioma proliferation and survival, in vitro and in vivo evidence, Mol. Med. Rep., 2014, 9: 229-234), bladder cancer (Wulfing, C., et al., Expression of the endothelin axis in bladder cancer: relationship to clinicopathologic parameters and long-term survival, Eur. Urol., 2005, 47(5), 593-600), head and neck cancer (Awano, S., et al., Endothelin system in oral squamous carcinoma cells: Specific siRNA targeting of ECE-1 blocks cell proliferation, Int. J. Cancer, 2006, 118, 1645- 1652), vulvar cancer (Eltze, E., et al., Expression and prognostic relevance of endothelin-B receptor in vulvar cancer, Oncology Rep., 2007, 18, 305-311), clear-cell renal cell carcinoma (Wuttig, D., et al., CD31, EDNRB and TSPAN7 are promising prognostic markers in clear- cell renal cell carcinoma revealed by genome-wide expression analyses of primary tumors and metastases, Int. J. Cancer, 2012, 131, E693-E704), multiple myeloma (Russignan, A., et al., Endothelin- 1 receptor blockade as a new possible therapeutic approach in multiple myeloma, British Journal of Haematology, 2017, 178, 781-793), pancreatic adenocarcinoma (Cook, N., et al., Endothelin- 1 and endothelin B receptor expression in pancreatic adenocarcinoma. J. Clin. Pathol., 2015, 68(4), 309-313), and Kaposi’s sarcoma (Rosano, R., et al., Endothelin receptor blockade inhibits molecular effectors of Kaposi's sarcoma cell invasion and tumor growth in vivo, Am. J. Pathology, 2003, 163(2), 753-762).

[00144] Without being bound by the theory, it is contemplated that in addition to tumor expression, ETBR is upregulated in the tumor microenvironment on the endothelial cells of tumor vasculature. For example, a method involving laser-capture microdissection was employed to conclusively demonstrate that ETBR is highly overexpressed in the surface of endothelial cells of the vasculature of ovarian cancer tumors and that ETBR overexpression was strongly correlated with low overall patient survival (Buckanovich, R.J., et al., Endothelin B receptor mediates the endothelial barrier to T cell homing to tumors and disables immune therapy, Nature Med., 2008; 14(1): 28-36). Expression of ETBR in tumor vasculature endothelial cells can reduce the level of intraepithelial tumor infiltrating leukocytes (TILs) in the tumor microenvironment via a mechanism that involved significant decrease of the intercellular adhesion molecule ICAM-1, which is required for leukocyte migration through the vasculature to the tumor (Buckanovich, R.J., et al., Endothelin B receptor mediates the endothelial barrier to T cell homing to tumors and disables immune therapy, Nature Med., 2008; 14(1): 28-36). Further, ETAR is required for the high expression of endothelial ICAM-1 and other adhesion molecules that are important for TIL migration (Coffman, L., et al., Endothelin receptor A is required for the recruitment of antitumor T cells and modulates chemotherapy induction of cancer stem cells. Cancer Biol. Ther., 2013, 14(2), 184-192). Thus, in some embodiments, a highly selective ETBR antagonist is used.

[00145] Moreover, neutrophils are involved in tumor progression and metastasis (Shaul, M.E., Fridlender, C.G., Tumour-associated neutrophils in patients with cancer, Nature Rev. Clin. Oncol., 2019, 16, 601-620) and ETBR antagonism can block pro-tumor neutrophil migration (Zarpelon, A.C., et al., Endothelin-1 induces neutrophil recruitment in adaptive inflammation via TNFa and CXCL1/CXCR2 in mice. Canadian J. Physiol, and Pharmacol., 2012, 90(2), 187-199). Without being bound by the theory, it is contemplated that ETBR antagonists can enhance the efficacy of immunotherapy drugs.

7.4 Compositions and Methods of Making the Same

7.4.1 Engineered Lasso Peptides

[00146] In a first aspect of the present disclosure, provided herein are engineered lasso peptides. Such engineered lasso peptides include variants of an endothelin receptor antagonistic lasso peptide described herein as SEQ ID NO: 1. These engineered lasso peptides include one or more amino acid alterations as compared to SEQ ID NO: 1. Such alterations include one or more specific amino acid substitutions, including combinations of substitutions as described herein. Moreover, the engineered lasso peptides described herein have improved properties and characteristics as compared to the parent lasso peptide of SEQ ID NO: 1. For example, when cyclized, the engineered lasso peptides described herein have higher specific binding affinity to ETBR (e.g., human and/or mouse), are more stable, have varied serum, plasma, or in vivo half-life, have varied in vivo exposure (AUC) and/or volume of distribution (Vz), and/or are more soluble in water or a mixture containing water (e.g., serum or plasma). Additionally, such engineered lasso peptides maintain or improve upon desirable therapeutic properties and characteristics of the parent lasso peptide of SEQ ID NO: 1. For example, the engineered lasso peptides described herein show no proteolysis or metabolism, have a clean safety profile, have low immunogenicity risk, have a long half-life (between 6 to 8 hours), high volume distribution into major organs frequently targeted for tumor treatment, and/or are selective for binding to ETBR as compared to ETAR.

[00147] In some embodiments, an engineered lasso peptide described herein allows for increasing the hydrogen bonding of the engineered lasso peptide to specific residues of ETBR (e.g., human and/or mouse ETBR) as compared to the parent lasso peptide of SEQ ID NO: 1. In some embodiments, an engineered lasso peptide described herein allows for increasing the hydrogen bonding interactions between the engineered lasso peptide and specific residues of human and/or mouse ETBR as compared to the parent lasso peptide of SEQ ID NO: 1. In some embodiments, an engineered lasso peptide described herein allows for increasing the hydrogen bonding interactions between the engineered lasso peptide and specific residues of human ETBR as compared to the parent lasso peptide of SEQ ID NO: 1. In some embodiments, an engineered lasso peptide described herein allows for increasing the hydrogen bonding interactions between the engineered lasso peptide and specific residues of mouse ETBR as compared to the parent lasso peptide of SEQ ID NO: 1. In some embodiments, an engineered lasso peptide described herein allows for increasing the hydrogen bonding interactions between the engineered lasso peptide and specific residues of human and mouse ETBR as compared to the parent lasso peptide of SEQ ID NO: 1. Such residues of ETBR include those found throughout the pocket of ETBR that bind endothelin ligands, including ET-1, ET-2, and ET-3, or in the receptor capping region of ETBR. The residues within the pocket of ETBR and the receptor capping region can be readily identified based on the activation mechanism of ETBR as described in Shihoya et al., Nature, 537 (7620):363-368 (2016) and X-ray and crystal structure of ETBR as described in Shihoya et al., Nat. Struct. Mol. Bio., 24(9):758-764 (2017) and Shihoya et al., Nature Commun., 9(1):4711 (2018), respectively. [00148] In some embodiments, engineered lasso peptides having variant amino acid sequences as compared to SEQ ID NO: 1 are described herein (e.g., Table 1). Table 1 summarizes the core peptide sequences of exemplary engineered lasso peptides provided herein, including one or more amino acid substitutions of the amino acid sequence of the engineered lasso peptide relative to SEQ ID NO: 1.

Table 1

[00149] In some embodiments, an engineered lasso peptide has a variant amino acid sequence relative to SEQ ID NO: 1, wherein the engineered lasso peptide includes one or more amino acid substitutions as described in Table 1. Accordingly, in some embodiments, the engineered lasso peptide includes one or more amino acid substitutions selected from the group consisting of N2A, N2D, N2S, W3A, W3D, W3E, W3Y, W3H, H4A, H4I, H4Q, H4M, H4L, H4W, H4Y, G5A, T6A, T6E, T6I, T6K, T6L, T6V, T6S, T6H, S7E, S7F, S7I,

S7L, S7N, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, W10A, F11Y, F11S, F11T, F11W, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, W16E, and W16K. In some embodiments, an engineered lasso peptide includes an N2A substitution. In some embodiments, an engineered lasso peptide includes an N2D substitution. In some embodiments, an engineered lasso peptide includes an N2S substitution. In some embodiments, an engineered lasso peptide includes an W3 A substitution. In some embodiments, an engineered lasso peptide includes an W3D substitution. In some embodiments, an engineered lasso peptide includes an W3E substitution. In some embodiments, an engineered lasso peptide includes an W3 Y substitution. In some embodiments, an engineered lasso peptide includes an W3H substitution. In some embodiments, an engineered lasso peptide includes an H4A substitution. In some embodiments, an engineered lasso peptide includes an H4I substitution. In some embodiments, an engineered lasso peptide includes an H4Q substitution. In some embodiments, an engineered lasso peptide includes an H4M substitution. In some embodiments, an engineered lasso peptide includes an H4L substitution. In some embodiments, an engineered lasso peptide includes an H4W substitution. In some embodiments, an engineered lasso peptide includes an H4Y substitution. In some embodiments, an engineered lasso peptide includes an G5A substitution. In some embodiments, an engineered lasso peptide includes an T6A substitution. In some embodiments, an engineered lasso peptide includes an T6E substitution. In some embodiments, an engineered lasso peptide includes an T6I substitution. In some embodiments, an engineered lasso peptide includes an T6K substitution. In some embodiments, an engineered lasso peptide includes an T6L substitution. In some embodiments, an engineered lasso peptide includes an T6V substitution. In some embodiments, an engineered lasso peptide includes an T6S substitution. In some embodiments, an engineered lasso peptide includes an T6H substitution. In some embodiments, an engineered lasso peptide includes an S7E substitution. In some embodiments, an engineered lasso peptide includes an S7F substitution. In some embodiments, an engineered lasso peptide includes an S7I substitution. In some embodiments, an engineered lasso peptide includes an S7L substitution. In some embodiments, an engineered lasso peptide includes an S7N substitution. In some embodiments, an engineered lasso peptide includes an S7W substitution. In some embodiments, an engineered lasso peptide includes an S7Y substitution. In some embodiments, an engineered lasso peptide includes an S7K substitution. In some embodiments, an engineered lasso peptide includes an S7R substitution. In some embodiments, an engineered lasso peptide includes an S7P substitution. In some embodiments, an engineered lasso peptide includes an P8F substitution. In some embodiments, an engineered lasso peptide includes an P8L substitution. In some embodiments, an engineered lasso peptide includes an W10A substitution. In some embodiments, an engineered lasso peptide includes an F11Y substitution. In some embodiments, an engineered lasso peptide includes an F11S substitution. In some embodiments, an engineered lasso peptide includes an F11T substitution. In some embodiments, an engineered lasso peptide includes an F11W substitution. In some embodiments, an engineered lasso peptide includes an F12H substitution. In some embodiments, an engineered lasso peptide includes an F12L substitution. In some embodiments, an engineered lasso peptide includes an F12M substitution. In some embodiments, an engineered lasso peptide includes an F12W substitution. In some embodiments, an engineered lasso peptide includes an F12Y substitution. In some embodiments, an engineered lasso peptide includes an N13S substitution. In some embodiments, an engineered lasso peptide includes an N13F substitution. In some embodiments, an engineered lasso peptide includes an N13H substitution. In some embodiments, an engineered lasso peptide includes an Y15F substitution. In some embodiments, an engineered lasso peptide includes an Y15L substitution. In some embodiments, an engineered lasso peptide includes an Y15H substitution. In some embodiments, an engineered lasso peptide includes an W16E substitution. In some embodiments, an engineered lasso peptide includes an W16K substitution.

[00150] In some embodiments, an engineered lasso peptide has a variant amino acid sequence of SEQ ID NO: 1, wherein the engineered lasso peptide includes two, three, four, five, or six amino acid substitutions as described in Table 1. Accordingly, in some embodiments, the engineered lasso peptide includes two, three, four, five, or six amino acid substitutions selected from the group consisting of N2A, N2D, N2S, W3A, W3D, W3E, W3Y, W3H, H4A, H4I, H4Q, H4M, H4L, H4W, H4Y, G5A, T6A, T6E, T6I, T6K, T6L, T6V, T6S, T6H, S7E, S7F, S7I, S7L, S7N, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, W10A, F11Y, F11S, F11T, F11W, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, W16E, and W16K. In some embodiments, the two amino acid substitutions are selected from the group consisting of: a) H4L and F11Y; b) H4M and F11Y; c) T6P and P8F; d) T6P and P8L; e) T6V and S7N; f) S7P and P8F; g) S7P and P8L; h) F11Y and F12H; and i) F11Y and F12Y. In some embodiments, the three amino acid substitutions are selected from the group consisting of: a) H4M, F11Y, and F12Y; b) W3E, H4M, and F11Y; c) W3Y, H4M, and F11Y; and d) W3H, H4M, and F11Y. In some embodiments, the four amino acid substitutions are selected from the group consisting of: a) W3D, H4M, F11Y, and F12Y; b) W3E, H4M, F11Y, and F12Y; c) W3Y, H4M, F11Y, and F12Y; and d) W3H, H4M, F11Y, and F12Y. In some embodiments, the five amino acid substitutions are selected from the group consisting of: a) W3D, H4M, F11Y, F12Y, and N13F; b) W3E, H4M, F11Y, F12Y, and N13F; c) W3Y, H4M, F11Y, F12Y, and N13F; d) W3H, H4M, F11Y, F12Y, and N13F; e) W3D, H4M, F11Y, F12Y, and N13H; f) W3E, H4M, F11Y, F12Y, and N13H; g) W3Y, H4M, F11Y, F12Y, and N13H; and h) W3H, H4M, F11Y, F12Y, and N13H. In some embodiments, the six amino acid substitutions are selected from the group consisting of: a) W3D, H4M, F11Y, F12Y, N13F, and S7I; b) W3E, H4M, F11Y, F12Y, N13F, and S7I; c) W3Y, H4M, F11Y, F12Y, N13F, and S7I; d) W3H, H4M, F11Y, F12Y, N13F, and S7I; e) W3D, H4M, F11Y, F12Y, N13H, and S7I; f) W3E, H4M, F11Y, F12Y, N13H, and S7I; g) W3Y, H4M, F11Y, F12Y, N13H, and S7I; h) W3H, H4M, F11Y, F12Y, N13H, and S7I; i) W3D, H4M, F11Y, F12Y, N13F, and S7Y; j) W3E, H4M, F11Y, F12Y, N13F, and S7Y; k) W3Y, H4M, F11Y, F12Y, N13F, and S7Y; 1) W3H, H4M, F11Y, F12Y, N13F, and S7Y; m) W3D, H4M, F11Y, F12Y, N13H, and S7Y; n) W3E, H4M, F11Y, F12Y, N13H, and S7Y; o) W3Y, H4M, F11Y, F12Y, N13H, and S7Y; p) W3H, H4M, F11Y, F12Y, N13H, and S7Y; q) W3D, H4M, F11Y, F12Y, N13F, and S7F; r) W3E, H4M, F11Y, F12Y, N13F, and S7F; s) W3Y, H4M, F11Y, F12Y, N13F, and S7F; t) W3H, H4M, F11Y, F12Y, N13F, and S7F; u) W3D, H4M, F11Y, F12Y, N13H, and S7F; v) W3E, H4M, F11Y, F12Y, N13H, and S7F; w) W3Y, H4M, F11Y, F12Y, N13H, and S7F; x) W3H, H4M, F11Y, F12Y, N13H, and S7F; y) W3D, H4M, F11Y, F12Y, N13F, and S7K; z) W3E, H4M, F11Y, F12Y, N13F, and S7K; aa) W3Y, H4M, F11Y, F12Y, N13F, and S7K; bb) W3H, H4M, F11Y, F12Y, N13F, and S7K; cc) W3D, H4M, F11Y, F12Y, N13H, and S7K; dd) W3E, H4M, F11Y, F12Y, N13H, and S7K; ee) W3Y, H4M, F11Y, F12Y, N13H, and S7K; ff) W3H, H4M, F11Y, F12Y, N13H, and S7K; gg) W3D, H4M, F11Y, F12Y, N13F, and S7R; hh) W3E, H4M, F11Y, F12Y, N13F, and S7R; ii) W3Y, H4M, F11Y, F12Y, N13F, and S7R; jj) W3H, H4M, F11Y, F12Y, N13F, and S7R; kk) W3D, H4M, F11Y, F12Y, N13H, and S7R; 11) W3E, H4M, F11Y, F12Y, N13H, and S7R; mm) W3Y, H4M, F11Y, F12Y, N13H, and S7R; and nn) W3H, H4M, F11Y, F12Y, N13H, and S7R.

[00151] In some embodiments, an engineered lasso peptide has a variant amino acid sequence of SEQ ID NO: 1, wherein the engineered lasso peptide has higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to the parent lasso peptide of SEQ ID NO: 1. Such an engineered lasso peptide includes one or more amino acid substitutions selected from the group consisting of F11Y, F12H, and F12Y. Accordingly, in some embodiments, an engineered lasso peptide has higher specific binding affinity to ETBR (e.g., human and/or mouse) and includes an F11Y substitution. In some embodiments, an engineered lasso peptide has higher specific binding affinity to ETBR (e.g., human and/or mouse) and includes an F12H substitution. In some embodiments, an engineered lasso peptide has higher specific binding affinity to ETBR (e.g., human and/or mouse) and includes an F12Y substitution.

[00152] In some embodiments, an engineered lasso peptide has a variant amino acid sequence relative to SEQ ID NO: 1, wherein the engineered lasso peptide has at least 1.5-fold higher specific binding affinity to ETBR (e.g, human and/or mouse) as compared to the parent lasso peptide of SEQ ID NO: 1. Accordingly, in some embodiments, an engineered lasso peptide having at least 1.5-fold, at least 2.5-fold, or at least 5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to the parent lasso peptide of SEQ ID NO: 1 includes an amino acid substitution selected from F11Y, F12H, and F12Y. Accordingly, in some embodiments, an engineered lasso peptide has at least 1.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) and includes an F11Y substitution. In some embodiments, an engineered lasso peptide has at least 2.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) and includes an F12H substitution. In some embodiments, an engineered lasso peptide has at least 5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) and includes an F12Y substitution. In addition to an amino acid substitution of F11Y, F12H, or F12Y, an engineered lasso peptide having at least 1.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to the parent lasso peptide of SEQ ID NO: 1 can include a further amino acid substitution as described herein. Thus, in some embodiments, an engineered lasso peptide having at least 1.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) has an amino acid substitution of F11Y and further includes a substitution selected from the group consisting of N2A, N2D, N2S, W3A, W3D, W3E, W3Y, W3H, H4A, H4I, H4Q, H4M, H4L, H4W, H4Y, G5A, T6A, T6E, T6I, T6K, T6L, T6V, T6S, T6H, S7E, S7F, S7I, S7L, S7N, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, W10A, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, W16E, and W16K. In some embodiments, an engineered lasso peptide having at least 1.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) has an amino acid substitution of F11Y and further includes a substitution selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, and W16K. In some embodiments, an engineered lasso peptide having at least 1.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) has an amino acid substitution of F11Y and further includes one, two, three, four, or five amino acid substitutions selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, and W16K. In some embodiments, an engineered lasso peptide having at least 1.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) has an amino acid substitution of F12H and further includes a substitution selected from the group consisting of N2A, N2D, N2S, W3A, W3D, W3E, W3Y, W3H, H4A, H4I, H4Q, H4M, H4L, H4W, H4Y, G5A, T6A, T6E, T6I, T6K, T6L, T6V, T6S, T6H, S7E, S7F, S7I, S7L, S7N, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, W10A, F11Y, F11 S, F11T, F11W, N13S, N13F, N13H, Y15F, Y15L, Y15H, W16E, and W16K. In some embodiments, an engineered lasso peptide having at least 1.5- fold higher specific binding affinity to ETBR (e.g., human and/or mouse) has an amino acid substitution of F12H and further includes a substitution selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F11Y, F11S, F11T, N13F, N13H, Y15F, Y15L, Y15H, and W16K. In some embodiments, an engineered lasso peptide having at least 1.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) has an amino acid substitution of F12H and further includes one, two, three, four, or five amino acid substitutions selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F11Y, F11S, F11T, N13F, N13H, Y15F, Y15L, Y15H, and W16K . In some embodiments, an engineered lasso peptide having at least 1.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) has an amino acid substitution of F12Y and further includes a substitution selected from the group consisting of N2A, N2D, N2S, W3A, W3D, W3E, W3Y, W3H, H4A, H4I, H4Q, H4M, H4L, H4W, H4Y, G5A, T6A, T6E, T6I, T6K, T6L, T6V, T6S, T6H, S7E, S7F, S7I, S7L, S7N, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, W10A, F11Y, F11 S, F11T, F11W, N13S, N13F, N13H, Y15F, Y15L, Y15H, W16E, and W16K. In some embodiments, an engineered lasso peptide having at least 1.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) has an amino acid substitution of F12Y and further includes a substitution selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F11Y, F11S, F11T, N13F, N13H, Y15F, Y15L, Y15H, and W16K. In some embodiments, an engineered lasso peptide having at least 1.5- fold higher specific binding affinity to ETBR (e.g., human and/or mouse) has an amino acid substitution of F12Y and further includes one, two, three, four, or five amino acid substitutions selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F11Y, F11S, F11T, N13F, N13H, Y15F, Y15L, Y15H, and W16K. In some embodiments, an engineered lasso peptide having at least 1.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) has two amino acid substitutions that are selected from the group consisting of: a) H4L and F11Y; b) H4M and F11Y; c) F11Y and F12H; and d) F11Y and F12Y. In some embodiments, an engineered lasso peptide having at least 1.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) has three amino acid substitutions that are selected from the group consisting of: a) H4M, F11Y, and F12Y; b) W3E, H4M, and F11Y; c) W3Y, H4M, and F11Y; and d) W3H, H4M, and F11Y.

In some embodiments, an engineered lasso peptide having at least 1.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) has four amino acid substitutions that are selected from the group consisting of: a) W3D, H4M, F11Y, and F12Y; b) W3E, H4M, F11Y, and F12Y; c) W3Y, H4M, F11Y, and F12Y; and d) W3H, H4M, F11Y, and F12Y. In some embodiments, an engineered lasso peptide having at least 1.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse) has five amino acid substitutions that are selected from the group consisting of: a) W3D, H4M, F11Y, F12Y, and N13F; b) W3E, H4M, F11Y, F12Y, and N13F; c) W3Y, H4M, F11Y, F12Y, and N13F; d) W3H, H4M, F11Y, F12Y, and N13F; e) W3D, H4M, F11Y, F12Y, and N13H; f) W3E, H4M, F11Y, F12Y, and N13H; g) W3Y, H4M, F11Y, F12Y, and N13H; and h) W3H, H4M, F11Y, F12Y, and N13H. In some embodiments, an engineered lasso peptide having at least 1.5-fold higher specific binding affinity to ETBR e.g., human and/or mouse) has six amino acid substitutions that are selected from the group consisting of: a) W3D, H4M, F11Y, F12Y, N13F, and S7I; b) W3E, H4M, F11Y, F12Y, N13F, and S7I; c) W3Y, H4M, F11Y, F12Y, N13F, and S7I; d) W3H, H4M, F11Y, F12Y, N13F, and S7I; e) W3D, H4M, F11Y, F12Y, N13H, and S7I; f) W3E, H4M, F11Y, F12Y, N13H, and S7I; g) W3Y, H4M, F11Y, F12Y, N13H, and S7I; h) W3H, H4M, F11Y, F12Y, N13H, and S7I; i) W3D, H4M, F11Y, F12Y, N13F, and S7Y; j) W3E, H4M, F11Y, F12Y, N13F, and S7Y; k) W3Y, H4M, F11Y, F12Y, N13F, and S7Y; 1) W3H, H4M, F11Y, F12Y, N13F, and S7Y; m) W3D, H4M, F11Y, F12Y, N13H, and S7Y; n) W3E, H4M, F11Y, F12Y, N13H, and S7Y; o) W3Y, H4M, F11Y, F12Y, N13H, and S7Y; p) W3H, H4M, F11Y, F12Y, N13H, and S7Y; q) W3D, H4M, F11Y, F12Y, N13F, and S7F; r) W3E, H4M, F11Y, F12Y, N13F, and S7F; s) W3Y, H4M, F11Y, F12Y, N13F, and S7F; t) W3H, H4M, F11Y, F12Y, N13F, and S7F; u) W3D, H4M, F11Y, F12Y, N13H, and S7F; v) W3E, H4M, F11Y, F12Y, N13H, and S7F; w) W3Y, H4M, F11Y, F12Y, N13H, and S7F; x) W3H, H4M, F11Y, F12Y, N13H, and S7F; y) W3D, H4M, F11Y, F12Y, N13F, and S7K; z) W3E, H4M, F11Y, F12Y, N13F, and S7K; aa) W3Y, H4M, F11Y, F12Y, N13F, and S7K; bb) W3H, H4M, F11Y, F12Y, N13F, and S7K; cc) W3D, H4M, F11Y, F12Y, N13H, and S7K; dd) W3E, H4M, F11Y, F12Y, N13H, and S7K; ee) W3Y, H4M, F11Y, F12Y, N13H, and S7K; ff) W3H, H4M, F11Y, F12Y, N13H, and S7K; gg) W3D, H4M, F11Y, F12Y, N13F, and S7R; hh) W3E, H4M, F11Y, F12Y, N13F, and S7R; ii) W3Y, H4M, F11Y, F12Y, N13F, and S7R; jj) W3H, H4M, F11Y, F12Y, N13F, and S7R; kk) W3D, H4M, F11Y, F12Y, N13H, and S7R; 11) W3E, H4M, F11Y, F12Y, N13H, and S7R; mm) W3Y, H4M, F11Y, F12Y, N13H, and S7R; and nn) W3H, H4M, F11Y, F12Y, N13H, and S7R.

[00153] In some embodiments, an engineered lasso peptide described herein, when cyclized, has higher specific binding affinity to ETBR (e.g., human and/or mouse), is more stable, and/or is more soluble in water or a mixture containing water (e.g., serum or plasma) as compared to the parent lasso peptide of SEQ ID NO: 1. In some embodiments, an engineered lasso peptide described herein, when cyclized, has higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to the parent lasso peptide of SEQ ID NO: 1. In some embodiments, an engineered lasso peptide described herein, when cyclized, is more stable. In some embodiments, an engineered lasso peptide described herein, when cyclized, is more soluble in water or a mixture containing water (e.g., serum or plasma)as compared to the parent lasso peptide of SEQ ID NO: 1. In some embodiments, an engineered lasso peptide described herein, when cyclized, has higher specific binding affinity to ETBR (e.g., human and/or mouse) and is more stable as compared to the parent lasso peptide of SEQ ID NO: 1. In some embodiments, an engineered lasso peptide described herein, when cyclized, has higher specific binding affinity to ETBR (e.g., human and/or mouse) and is more soluble under in water or a mixture containing water (e.g., serum or plasma) as compared to the parent lasso peptide of SEQ ID NO: 1. In some embodiments, an engineered lasso peptide described herein, when cyclized, is more stable and is more soluble in water or a mixture containing water (e.g., serum or plasma) as compared to the parent lasso peptide of SEQ ID NO: 1.

[00154] In some embodiments, an engineered lasso peptide having higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to parent lasso peptide of SEQ ID NO: 1 has at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold higher specific binding affinity to ETBR (e.g., human and/or mouse). In some embodiments, an engineered lasso peptide having higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to parent lasso peptide of SEQ ID NO: 1 has at least 10% higher specific binding affinity to ETBR (e.g., human and/or mouse). In some embodiments, an engineered lasso peptide having higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to parent lasso peptide of SEQ ID NO: 1 has at least 50% higher specific binding affinity to ETBR (e.g., human and/or mouse). In some embodiments, an engineered lasso peptide having higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to parent lasso peptide of SEQ ID NO: 1 has at least 100% higher specific binding affinity to ETBR (e.g., human and/or mouse). In some embodiments, an engineered lasso peptide having higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to parent lasso peptide of SEQ ID NO: 1 has at least 1.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse). In some embodiments, an engineered lasso peptide having higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to parent lasso peptide of SEQ ID NO: 1 has at least 2-fold higher specific binding affinity to ETBR (e.g., human and/or mouse). In some embodiments, an engineered lasso peptide having higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to parent lasso peptide of SEQ ID NO: 1 has at least 2.5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse). In some embodiments, an engineered lasso peptide having higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to parent lasso peptide of SEQ ID NO: 1 has at least 5-fold higher specific binding affinity to ETBR (e.g., human and/or mouse). In some embodiments, an engineered lasso peptide having higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to parent lasso peptide of SEQ ID NO: 1 has at least 10-fold higher specific binding affinity to ETBR (e.g., human and/or mouse). In some embodiments, an engineered lasso peptide having higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to parent lasso peptide of SEQ ID NO: 1 has at least 20-fold higher specific binding affinity to ETBR (e.g., human and/or mouse). In some embodiments, an engineered lasso peptide having higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to parent lasso peptide of SEQ ID NO: 1 has at least 50-fold higher specific binding affinity to ETBR (e.g., human and/or mouse). In some embodiments, an engineered lasso peptide having higher specific binding affinity to ETBR (e.g., human and/or mouse) as compared to parent lasso peptide of SEQ ID NO: 1 has at least 100-fold higher specific binding affinity to ETBR (e.g., human and/or mouse).

[00155] In some embodiments, an engineered lasso peptide described herein preferentially bind to ETBR over ETAR. In some embodiments, the engineered lasso peptide preferentially binds to ETBR with a K_D or Ki that is about 75%, about 50%, about 25%, about 10%, about 5%, about 2.5%, or about 1% of the K_D or Ki exhibited relative to binding of ETAR. In some embodiments, the ratio between the K_D or Ki exhibited by the engineered lasso peptide when binding to the ETAR and the K_D or Ki exhibited when binding to ETBR is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 100 fold, at least 500 fold, at least 1,000 fold, at least 1,500 fold, at least 2,000 fold, at least 2,500 fold, at least 5,000 fold, at least 10⁴ fold, or at least 10⁵ fold. In some embodiments, the engineered lasso peptide specifically binds to ETBR and does not exhibit detectable binding to ETAR. [00156] In some embodiments, an engineered lasso peptide having higher stability as compared to parent lasso peptide of SEQ ID NO: 1 is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold more stable. In some embodiments, such stability is measured by thermal degradation. For example, thermal degradation can be determined by heating the engineered lasso peptide in a solvent, such as water, at temperatures up to 100°C, and measuring the engineered lasso peptide remaining over time by high-performance liquid chromatograph (HPLC) or liquid chromatography- mass spectrometry (LCMS). In some embodiments, such stability is measured by proteolytic degradation through hydrolysis of a peptide bond. For example, proteolytic degradation is typically measured by adding an engineered lasso peptide in water to human or animal serum or plasma and measuring engineered lasso peptide remaining over time by HPLC or LCMS. Similarly for specific proteases, engineered lasso peptides can be added to solutions of a protease of interest (e.g., elastase, carboxypeptidase Y, trypsin, or chymotrypsin) and allowed to incubate, with engineered lasso peptide remaining measured over time by HPLC or LCMS. [00157] In some embodiments, an engineered lasso peptide having more solubility as compared to parent lasso peptide of SEQ ID NO: 1 is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold more soluble in water or a mixture containing water (e.g., serum or plasma). In some embodiments, such solubility is measured by dissolving the engineered lasso peptide (i.e., the solute) in water up to saturation, at which point the mg of engineered lasso peptide per mL water (mg/mL) is the measure of solubility. Alternatively, a saturated solution of engineered lasso peptides is filtered or centrifuged, and the concentration of the soluble fraction is quantified by HPLC or LCMS. [00158] In some embodiments, an engineered lasso peptide comprises an amino acid sequence of an engineered lasso peptide depicted in Table 1. Accordingly, in some embodiments, the engineered lasso peptide comprises an amino acid sequence selected from SEQ ID NOS: 2 to 117.

[00159] In some embodiments, an engineered lasso peptide consists essentially of an amino acid sequence of an engineered lasso peptide depicted in Table 1. Accordingly, in some embodiments, the engineered lasso peptide consists essentially of an amino acid sequence selected from SEQ ID NOS: 2 to 117.

[00160] In some embodiments, an engineered lasso peptide consists of an amino acid sequence of an engineered lasso peptide depicted in Table 1. Accordingly, in some embodiments, the engineered lasso peptide consists of an amino acid sequence selected from SEQ ID NOS: 2 to 117.

[00161] In some embodiments, an engineered lasso peptide described herein further includes a leader sequence. For example, in some embodiments, provided herein is an engineered lasso peptide having a variant amino acid sequence of SEQ ID NO: 1 and a leader sequence, thereby resulting in a lasso precursor peptide. Accordingly, provide herein is a lasso precursor peptide having a variant amino acid sequence of SEQ ID NO: 1 as described herein and a leader sequence. An exemplary leader sequence useful for generating such a lasso precursor peptide include the amino acid sequence of MTEQSEQTPTEYIPPMLVEVGEFTEDTL (SEQ ID NO: 118). Additional leader sequences are known in the art, such as those disclosed in PCT application publication number WO2019/191571 and Tietz et al. (2017) Nature Chemical Biology, 13.

10.1038/nchembio.2319, which are incorporated herein by reference in their entirety. Such leader sequences are recognized by lasso peptide biosynthesis component proteins, and hence lasso precursor peptides as described herein can be processed by the same lasso peptide biosynthesis component proteins into matured engineered lasso peptides having different amino acid sequences, including the variant amino acid sequences of SEQ ID NO: 1 as described herein. Therefore, in some embodiments, to produce an engineered lasso peptide as described herein, the core peptide sequences (e.g., sequence of Table 1) can be fused to any known leader sequence, which when processed by lasso peptide biosynthesis component proteins, produces an engineered lasso peptide as described herein.

[00162] In some embodiments, an engineered lasso peptide described herein further includes a heterologous amino acid sequence fused to the tail of an engineered lasso peptide described herein, including a parent lasso peptide of SEQ ID NO: 1 or an engineered lasso peptide. For example, in some embodiments, provided herein is an engineered lasso peptide having an amino acid sequence described in Table 1 and an amino acid sequence of a heterologous protein (e.g., a fusion partner), thereby resulting in a fusion protein between the engineered lasso peptide and the heterologous sequence. Accordingly, provided herein is an engineered lasso peptide having a variant amino acid sequence of SEQ ID NO: 1 as described herein and a fusion partner. Also provided herein is a lasso peptide having the amino acid sequence of SEQ ID NO: 1 as described herein and a fusion partner.

[00163] In addition to the amino acid substitutions described in Table 1 and the combinations thereof, an engineered lasso peptide provided herein can have a further alteration of one or more amino acid residues. For example, in some embodiments, the engineered lasso peptide further includes a C-terminal tryptophan (W) modification. Such modifications of the C-terminal W of the engineered lasso peptide include a modification selected from the group consisting of: a) tryptophan having a C-terminal methyl ester group (-CCEMe) in place of the carboxylic acid group (-CO2H) (W-OMe); b) tryptophan having a C-terminal benzyl ester group (-CO2Bn) in place of the carboxylic acid group (-CO2H) (W- OBn); c) tryptophan having a C-terminal amide group (-CONH2) in place of the carboxylic acid group (-CO2H) (W-NH2); d) 7-hydroxyl-trptophan (W-7-OH); e) 2-naphthylalanine (Nal) in place of W; and f) an aza derivative of tryptophan - (2S)-2-amino-3-(lH-pyrrolo[5,4- b]pyri din-3 -yl)propanoic acid - in place of W having the structure of:

Accordingly, in some embodiments, the engineered lasso peptide described herein further includes a C-terminal W-OMe. In some embodiments, the engineered lasso peptide described herein further includes a C-terminal W-OBn. In some embodiments, the engineered lasso peptide described herein further includes a C-terminal W-NH2. In some embodiments, the engineered lasso peptide described herein further includes a C-terminal W- 7-OH. In some embodiments, the engineered lasso peptide described herein further includes a C-terminal Nal. In some embodiments, the engineered lasso peptide described herein further includes a C-terminal Trn.

[00164] Additionally, an engineered lasso peptide provided herein having the amino acid substitutions described in Table 1 and the combinations thereof can have a post-translational modification. For example, amino acids that contain functionality, such as amino or hydroxyl groups, can be modified by phosphorylation, acylation, methylation, esterification, prenylation, hydroxylation, and glycosylation of an engineered lasso peptide described herein. In addition, a cysteine can be introduced into an engineered lasso peptide described herein and subsequently chemically reacted with alkyl or aryl halides or reacted with activated double bonds to form S-alkylated derivatives of an engineered lasso peptide provided herein.

[00165] In some embodiments, an engineered lasso peptide provided herein has an amino acid sequence that is a variant of SEQ ID NO: 1 that includes one or more substitutions as described in Table 1 and/or a combination of substitutions described in Table 1, wherein the portion, other than the one or more substitutions described in Table 1 or the combination of substitutions described in Table 1, of the engineered lasso peptide has one, two, three, four, five, or six additional alterations relative to SEQ ID NO: 1, or is identical to the amino acid sequence of SEQ ID NO: 1. Accordingly, in some embodiments, an engineered lasso peptide provided herein has an amino acid sequence that includes one or more substitutions as described in Table 1 and/or a combination of substitutions described in Table 1, and the portion, other than the substitution described in Table 1 or the combination of substitutions described in Table 1, of the engineered lasso peptide has one, two, three, four, five, or six additional alterations relative to SEQ ID NO: 1. In some embodiments, an engineered lasso peptide provided herein has an amino acid sequence that includes one or more substitutions as described in Table 1 and/or a combination of substitutions described in Table 1, and the portion, other than the substitution described in Table 1 or the combination of substitutions described in Table 1, of the engineered lasso peptide has two additional alterations relative to SEQ ID NO: 1. In some embodiments, an engineered lasso peptide provided herein has an amino acid sequence that includes one or more substitutions as described in Table 1 and/or a combination of substitutions described in Table 1, and the portion, other than the substitution described in Table 1 or the combination of substitutions described in Table 1, of the engineered lasso peptide has three additional alterations relative to SEQ ID NO: 1. In some embodiments, an engineered lasso peptide provided herein has an amino acid sequence that includes one or more substitutions as described in Table 1 and/or a combination of substitutions described in Table 1, and the portion, other than the substitution described in Table 1 or the combination of substitutions described in Table 1, of the engineered lasso peptide has four additional alterations relative to SEQ ID NO: 1. In some embodiments, an engineered lasso peptide provided herein has an amino acid sequence that includes one or more substitutions as described in Table 1 and/or a combination of substitutions described in Table 1, and the portion, other than the substitution described in Table 1 or the combination of substitutions described in Table 1, of the engineered lasso peptide has five additional alterations relative to SEQ ID NO: 1. In some embodiments, an engineered lasso peptide provided herein has an amino acid sequence that includes one or more substitutions as described in Table 1 and/or a combination of substitutions described in Table 1, and the portion, other than the substitution described in Table 1 or the combination of substitutions described in Table 1, of the engineered lasso peptide has six additional alterations relative to SEQ ID NO: 1. In some embodiments, an engineered lasso peptide provided herein has an amino acid sequence that includes one or more substitutions as described in Table 1 and/or a combination of substitutions described in Table 1, and the portion, other than the substitution described in Table 1 or the combination of substitutions described in Table 1, of the engineered lasso peptide is identical to SEQ ID NO: 1.

[00166] In some embodiments, an engineered lasso peptide provided herein differs from its respective core peptide having an amino acid sequence as described herein in that the engineered lasso peptide has a lariat conformation (e.g., is cyclized), while the core peptide itself does not necessarily possess such a secondary structure. Accordingly, in some embodiments, an engineered lasso peptide described herein is G1-D9 cyclized. In some embodiments, an engineered lasso peptide comprises an amino acid sequence depicted in Table 1, and possesses the lariat conformation through G1-D9 cyclization. In some embodiments, the engineered lasso peptide consists essentially of an amino acid sequence depicted in Table 1, and possesses the lariat conformation through G1-D9 cyclization. In some embodiments, the engineered lasso peptide consists of an amino acid sequence depicted in Table 1, and possesses the lariat conformation through G1-D9 cyclization.

[00167] In some embodiments, an engineered lasso peptide described herein competes with endothelin for binding with ETBR, wherein the endothelin is selected from ET-1, ET-2 or ET-3. In some embodiments, an engineered lasso peptide described herein competes with ET-1 for binding with ETBR. In some embodiments, an engineered lasso peptide described herein competes with ET-2 for binding with ETBR. In some embodiments, an engineered lasso peptide described herein competes with ET-3 for binding with ETBR.

[00168] In some embodiments, an engineered lasso peptide described herein preferentially binds a target molecule with a K_D or Ki less than half of the K_D or Ki exhibited relative to the reference molecule. In some embodiments, an engineered lasso peptide described herein preferentially binds a target molecule with a K_D or Ki at least 10 times less than the K_D or Ki exhibited relative to the reference molecule. In some embodiments, an engineered lasso peptide described herein preferentially binds a target molecule with a K_D or Ki that is about 75%, about 50%, about 25%, about 10%, about 5%, about 2.5%, or about 1% of the K_D or Ki exhibited relative to the reference molecule. In some embodiments, the ratio between the K_D or Ki exhibited by the engineered lasso peptide when binding to the reference molecule and the K_D or Ki exhibited when binding to the target molecule is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 100 fold, at least 500 fold, at least 10³ fold, at least 10⁴ fold, or at least 10⁵ fold less.

[00169] In some embodiments, an engineered lasso peptide described herein preferentially binds to ETBR over ETAR. In some embodiments, an engineered lasso peptide preferentially binds to ETBR with a K_D or Ki to ETBR that is about 75%, about 50%, about 25%, about 10%, about 5%, about 2.5%, or about 1% of the K_D or Ki exhibited relative to binding of ETAR. In some embodiments, the ratio between the K_D or Ki exhibited by the engineered lasso peptide when binding to the ETBR and the K_D or Ki exhibited when binding to ETAR is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 100 fold, at least 500 fold, at least 10³ fold, at least 10⁴ fold, or at least 10⁵ fold lower. In some embodiments, an engineered lasso specifically binds to ETBR and does not exhibit detectable binding to ETAR. In some embodiments, an engineered lasso specifically binds to ETBR and do not exhibit detectable binding to ETAR.

[00170] In some embodiments, an engineered lasso peptide described herein specifically antagonizes ETBR over ETAR. In some embodiments, the engineered lasso specifically antagonizes ETBR with an IC₅₀ less than half of the IC₅₀ exhibited relative to ETAR. In some embodiments, the engineered lasso peptide specifically antagonizes ETBR with an IC₅₀ to ETBR that is about 75%, about 50%, about 25%, about 10%, about 5%, about 2.5%, or about 1% of the IC₅₀ exhibited relative to the ETAR. In some embodiments, the ratio between the IC₅₀ exhibited by the engineered lasso peptide with respect to the ETBR and the IC₅₀ exhibited with respect to the ETAR is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 100 fold, at least 500 fold, at least 10³ fold, at least 10⁴ fold, or at least 10⁵ fold lower. In some embodiments, the engineered lasso peptide described herein specifically antagonizes ETBR and does not exhibit detectable inhibition or attenuation of ETAR.

[00171] In some embodiments, an engineered lasso peptide described herein preferentially binds to endothelin B receptor- 1 (ETBR1) over endothelin B receptor-2 (ETBR2). In some embodiments, an engineered lasso peptide preferentially binds to ETBR1 with a K_D or Ki to ETBR1 that is about 75%, about 50%, about 25%, about 10%, about 5%, about 2.5%, or about 1% of the K_D or Ki exhibited relative to binding of ETBR2. In some embodiments, the ratio between the K_D or Ki exhibited by the engineered lasso peptide when binding to the ETBR1 and the K_D or Ki exhibited when binding to ETBR2 is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 100 fold, at least 500 fold, at least 10³ fold, at least 10⁴ fold, or at least 10⁵ fold lower. In some embodiments, an engineered lasso peptide specifically binds to ETBR1 and does not exhibit detectable binding to ETBR2.

[00172] In some embodiments, an engineered lasso peptide described herein specifically antagonizes ETBR1. In some embodiments, an engineered lasso peptide specifically antagonizes ETBR1 over ETBR2. In some embodiments, the engineered lasso peptide specifically antagonizes ETBR with an IC₅₀ less than half of the IC₅₀ exhibited relative to ETAR. In some embodiments, the engineered lasso peptide specifically antagonizes ETBR1 with an IC₅₀ to ETBR1 that is about 75%, about 50%, about 25%, about 10%, about 5%, about 2.5%, or about 1% of the IC₅₀ exhibited relative to the ETBR2. In some embodiments, the ratio between the IC₅₀ exhibited by the engineered lasso peptide with respect to the ETBR1 and the IC50 exhibited with respect to the ETBR2 is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 100 fold, at least 500 fold, at least 10³ fold, at least 10⁴ fold, or at least 10⁵ fold less. In some embodiments, the engineered lasso peptide specifically antagonizes ETBR1 and does not exhibit detectable antagonization of ETBR2.

[00173] In some embodiments, an engineered lasso peptide that specifically binds to a target molecule has a K_D of less than or equal to 100 μM, 80 μM, 50 μM, 25 μM, 10 μM, 5 μM, 1 μM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM. In some embodiments, an engineered lasso peptide that specifically antagonizes to a target molecule has a Ki of less than or equal to 100 μM, 80 μM, 50 μM, 25 μM, 10 μM, 5 μM, 1 μM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM.

[00174] In some embodiment, an engineered lasso peptide described herein is said to specifically bind to a target molecule, for example, when the K_D is ≤10^-6 M. In some embodiments, an engineered lasso peptide specifically binds to a target molecule with a K_D of from about 10^-6 M to about 10^-12 M. In some embodiments, an engineered lasso peptide specifically binds to a target molecule with high affinity when the K_D is ≤10^-8 M or K_D is <10^-9 M. In some embodiments, an engineered lasso peptide specifically binds to a purified human target molecule with a K_D of from 1 x 10^-9 M to 10 x 10^-9 M as measured by Biacore®. In some embodiments, an engineered lasso peptide specifically binds to a purified human target molecule with a K_D of from 0.1 x 10^-9 M to 1 x 10^-9 M as measured by KinExA™ (Sapidyne, Boise, ID). In some embodiments, an engineered lasso peptide specifically binds to a target molecule expressed on cells with a K_D of from 0.1 x 10^-9 M to 10 x 10^-9 M. In some embodiments, an engineered lasso peptide specifically binds to a human target molecule expressed on cells with a K_D of from 0.1 x 10^-9 M to 1 x 10^-9 M. In some embodiments, an engineered lasso peptide specifically binds to a human target molecule expressed on cells with a K_D of 1 x 10^-9 M to 10 x 10^-9 M. In some embodiments, an engineered lasso peptide specifically binds to a human target molecule expressed on cells with a K_D of about 0.1 x 10^-9 M , about 0.5 x 10^-9 M, about 1 x 10^-9 M, about 5 x 10^-9 M, about 10 x 10^-9 M, or any range or interval thereof. In some embodiments, the engineered lasso peptides specifically bind to a non-human target molecule (e.g., a mouse ETBR) expressed on cells with a K_D of 0.1 X 10^-9 M to 10 X 10^-9 M. In some embodiments, the engineered lasso peptides specifically bind to a non-human target molecule (e.g., a mouse ETBR) expressed on cells with a K_D of from 0.1 x 10^-9 M to 1 x 10^-9 M. In some embodiments, the engineered lasso peptides specifically bind to a non-human target molecule (e.g., a mouse ETBR) expressed on cells with a K_D of 1 x 10^-9 M to 10 x 10^-9 M. In some embodiments, the engineered lasso peptides specifically bind to a non-human target molecule (e.g., a mouse ETBR) expressed on cells with a K_D of about 0.1 x 10^-9 M, about 0.5 x 10^-9 M, about 1 x 10^-9 M, about 5 x 10^-9 M, about 10 x 10^-9 M, or any range or interval thereof. [00175] In some embodiments, an engineered lasso peptide provided herein that binds ETBR is one that binds ETBR with sufficient affinity such that the engineered lasso peptide is useful, for example, as a diagnostic or therapeutic agent in targeting a cell or tissue expressing ETBR, and does not significantly cross-react with other molecules (e.g., ETAR). In such embodiments, the extent of binding of the engineered lasso peptide to a non-target molecule will be less than about 10% of the binding of the engineered lasso peptide to its particular target molecule, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA.

[00176] In some embodiments, an engineered lasso peptide provided herein, upon binding to an ETBR, inhibits the ETBR. In some embodiments, an engineered lasso peptide provided herein antagonizes at least one ETBR-mediated signaling pathway. In some embodiments, the inhibition of ETBR-mediated signaling pathway is measured by (a) inhibition of release of relaxing factors; (b) upregulation of intercellular adhesion molecule-1 (ICAM-1) expression and clustering; (c) increasing in migration of intraepithelial tumor infiltrating leukocytes (TILs) into the microenvironment of the neoplastic cells; (d) inhibition of angiogenesis in the microenvironment of neoplastic cells; (e) inhibition of growth and/or metastasis of neoplastic cells; (f) increasing in apoptosis of neoplastic cells; or any combination of (a) to (f). In some embodiments, the relaxing factors are selected from nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor, Ca2+, protein kinase C, mitogen-activated protein kinase, or any combination thereof. In some embodiments, the TILs comprises neutrophils, T cells, B cells, NK cells, monocytes or a combination thereof. In some embodiments, the monocytes include macrophages and/or dendritic cells. In some embodiments, the any of the above activities (a) to (f) is inhibited at least 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99% or 100%.

[00177] In some embodiments, the inhibition of at least one ETBR-mediated signaling pathway occurs simultaneously as the reduction of ETBR levels as described herein. In some embodiments, the inhibition of at least one ETBR-mediated signaling pathway occurs before the reduction of ETBR levels. In some embodiments, the inhibition of at least one ETBR- mediated signaling pathway occurs after the reduction of ETBR levels. In some embodiments, the reduction of ETBR levels occurs about 1 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 2 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 3 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 4 hour after the inhibition of the at least one ETBR- mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 5 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 10 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 12 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 24 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 36 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 48 hour after the inhibition of the at least one ETBR-mediated signaling pathway.

[00178] In some embodiments, an engineered lasso peptide provided herein, upon binding to ETBR, antagonizes at least one ETBR-mediated signaling pathway by at least %, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99% or 100%. In some embodiments, an engineered lasso peptide provided herein reduces ETBR levels on the surface of neoplastic cells and/or endothelial cells in the microenvironment of the neoplastic cells due to ligand-induced ETBR internalization. In some embodiments, such reduction in ETBR levels is by at least 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99% or 100%. In some embodiments, an engineered lasso peptide provided herein downregulates ETBR expression on the surface of the neoplastic cells and/or endothelial cells in the microenvironment of the neoplastic cells. In some embodiments, an engineered lasso peptide provided herein downregulates ETBR expression on the surface of neoplastic cells produced by the proliferative disease. In some embodiments, an engineered lasso peptide provided herein downregulates ETBR expression on endothelial cells in the microenvironment of the neoplastic cells produced by the proliferative disease. In some embodiments, an engineered lasso peptide provided herein, upon binding to ETBR, reduce ETBR levels by at least 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99% or 100% and/or down regulates ETBR expression by at least 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99% or 100%.

7.4.2 Nucleic acids

[00179] In a related aspect of the present disclosure, provided herein are recombinant nucleic acids encoding an engineered lasso peptide described herein. Accordingly, in some embodiments, provided herein is a recombinant nucleic acid that has a nucleotide sequence encoding engineered lasso peptides having variant amino acid sequences as compared to SEQ ID NO: 1. Accordingly, in some embodiments, provided herein is a recombinant nucleic acid selected from (a) a nucleic acid molecule encoding an engineered lasso peptide having a variant amino acid sequence of SEQ ID NO: 1, wherein the engineered lasso peptide includes one or more amino acid substitutions as described in Table 1; (b) a recombinant nucleic acid that hybridizes to an isolated nucleic acid of (a) under highly stringent hybridization conditions; and (c) a recombinant nucleic acid that is complementary to (a) or (b).

[00180] In some embodiments, provided herein is a recombinant nucleic acid encoding an engineered lasso peptide having one or more amino acid substitutions selected from the group consisting of N2A, N2D, N2S, W3A, W3D, W3E, W3Y, W3H, H4A, H4I, H4Q, H4M, H4L, H4W, H4Y, G5A, T6A, T6E, T6I, T6K, T6L, T6V, T6S, T6H, S7E, S7F, S7I, S7L, S7N, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, W10A, F11Y, F11S, F11T, F11W, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, W16E, and W16K. In some embodiments, the recombinant nucleic acid encodes an engineered lasso peptide having a variant amino acid sequence of SEQ ID NO: 1, wherein the engineered lasso peptide includes two, three, four, five, or six amino acid substitutions as described in Table 1. In some embodiments, the recombinant nucleic acid encodes an engineered lasso peptide having a variant amino acid sequence of SEQ ID NO: 1, wherein the engineered lasso peptide has higher specific binding affinity to ETBR as compared to the parent lasso peptide of SEQ ID NO: 1 and includes one or more amino acid substitutions selected from the group consisting of F11Y, F12H, and F12Y. In some embodiments, the recombinant nucleic acid encodes an engineered lasso peptide having a variant amino acid sequence of SEQ ID NO: 1, wherein the engineered lasso peptide has at least 1.5-fold higher specific binding affinity to ETBR as compared to the parent lasso peptide of SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered lasso peptide that preferentially bind to ETBR over ETAR. In some embodiments, the recombinant nucleic acid encodes an engineered lasso peptide having higher stability as compared to parent lasso peptide of SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered lasso peptide having more solubility as compared to parent lasso peptide of SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered lasso peptide comprising an amino acid sequence of an engineered lasso peptide depicted in Table 1. In some embodiments, the recombinant nucleic acid comprises a nucleotide sequence selected from SEQ ID NO: 119-235 encoding the engineered lasso peptide. In some embodiments, the recombinant nucleic acid encodes an engineered lasso peptide consisting essentially of an amino acid sequence of an engineered lasso peptide depicted in Table 1. In some embodiments, the recombinant nucleic acid encodes an engineered lasso peptide consisting of an amino acid sequence of an engineered lasso peptide depicted in Table 1. In some embodiments, the recombinant nucleic acid encodes an engineered lasso peptide having, in addition to one or more amino acid substitutions described in Table 1 or the combinations thereof, a further alteration of one or more amino acid residues. In some embodiments, the recombinant nucleic acid encodes an engineered lasso peptide provided herein having an amino acid sequence that is a variant of SEQ ID NO: 1 that includes one or more substitutions as described in Table 1 and/or a combination of substitutions described in Table 1, wherein the portion, other than the one or more substitutions described in Table 1 or the combination of substitutions described in Table 1, of the engineered lasso peptide has one, two, three, four, five, or six additional alterations relative to SEQ ID NO: 1, or is identical to the amino acid sequence of SEQ ID NO: 1.

[00181] In some embodiments, the recombinant nucleic acid encodes an engineered lasso peptide having a leader sequence. For example, in some embodiments, provided herein is a recombinant nucleic acid encoding an engineered lasso peptide having a variant amino acid sequence of SEQ ID NO: 1 and a leader sequence, thereby resulting in a lasso precursor peptide when transcribed and translated. Accordingly, provide herein is a recombinant nucleic acid encoding a lasso precursor peptide having a variant amino acid sequence of SEQ ID NO: 1 as described herein and a leader sequence. An exemplary leader sequence useful for generating such a lasso precursor peptide include the amino acid sequence of MTEQSEQTPTEYIPPMLVEVGEFTEDTL (SEQ ID NO: 118). Accordingly, in some embodiments, provided herein is a recombinant nucleic acid encoding a lasso precursor peptide having an amino acid sequence select from SEQ ID NOS: 238-363. In some embodiments, the recombinant nucleic acid comprises a nucleotide sequence selected from SEQ ID NOS: 364-480 encoding the lasso precursor peptide. Additional leader sequences are known in the art, such as those disclosed in PCT application publication number WO2019/191571 and Tietz et al. (2017) Nat Chem Bio, 13. 10. 1038/nchembio.2319, which are incorporated herein by reference in their entirety.

[00182] In some embodiments, provided herein is a recombinant nucleic acid that hybridizes under highly stringent hybridization conditions to an isolated nucleic acid encoding an engineered lasso peptide described herein. Accordingly, in some embodiments, the recombinant nucleic acid is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered lasso peptide that is a variant of a parent lasso peptide (SEQ ID NO: 1), such as an engineered lasso peptide having one or more substitutions at a position described in Table 1, and, in some embodiments, a combination of substitutions described in Table 1. In some embodiments, the recombinant nucleic acid molecule is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered lasso peptide having one or more substitutions at a position described in Table 1. In some embodiments, the recombinant nucleic acid is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered lasso peptide having a combination of substitutions described in Table 1.

[00183] A recombinant nucleic acid encoding an engineered lasso peptide described herein also includes a nucleic acid that hybridizes to a nucleic acid disclosed herein or a nucleic acid that hybridizes to a nucleic acid that encodes an amino acid sequence disclosed.

Hybridization conditions can include highly stringent, moderately stringent, or low stringency hybridization conditions that are well known to one of skill in the art such as those described herein. Similarly, a recombinant nucleic acid that can be used in the compositions and methods described herein can be described as having a certain percent sequence identity to a nucleic acid disclosed herein or a nucleic acid that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein. For example, the nucleic acid can have at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or be identical, to a nucleotide described herein.

[00184] Stringent hybridization refers to conditions under which hybridized polynucleotides are stable. As known to those of skill in the art, the stability of hybridized polynucleotides is reflected in the melting temperature (Tm) of the hybrids. In general, the stability of hybridized polynucleotides is a function of the salt concentration, for example, the sodium ion concentration, and temperature. A hybridization reaction can be performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Reference to hybridization stringency relates to such washing conditions. Highly stringent hybridization includes conditions that permit hybridization of only those nucleotide sequences that form stable hybridized polynucleotides in 0.018M NaCl at 65°C, for example, if a hybrid is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.1X SSPE, and 0.1% SDS at 65°C. Hybridization conditions other than highly stringent hybridization conditions can also be used to describe the nucleotide sequences disclosed herein. For example, the phrase moderately stringent hybridization refers to conditions equivalent to hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.2X SSPE, 0.2% SDS, at 42°C. The phrase low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5X Denhart's solution, 6X SSPE, 0.2% SDS at 22°C, followed by washing in IX SSPE, 0.2% SDS, at 37°C. Denhart's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 20X SSPE (sodium chloride, sodium phosphate, ethylene diamine tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other suitable low, moderate and high stringency hybridization buffers and conditions are well known to those of skill in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

[00185] A recombinant nucleic acid encoding an engineered lasso peptide described herein can have at least a certain sequence identity to a nucleotide sequence disclosed herein. Accordingly, in some aspects described herein, a recombinant nucleic acid encoding an engineered lasso peptide has a nucleotide sequence of at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, or is identical, to a nucleic acid disclosed herein or a nucleic acid that hybridizes to a nucleic acid that encodes an amino acid sequence disclosed herein. [00186] It is understood that a recombinant nucleic acid described herein or an engineered lasso peptide described here can exclude a wild-type or parental sequence, for example, a parental sequence, such as SEQ ID NO: 1. One skilled in the art will readily understand the meaning of a parental or wild-type sequence based on what is well known in the art. It is further understood that such a recombinant nucleic acid described herein can exclude a nucleotide sequence encoding a naturally occurring amino acid sequence as found in nature. Similarly, an engineered lasso peptide described herein can exclude an amino acid sequence as found in nature. Thus, in some embodiments, the recombinant nucleic acid or engineered lasso peptide described herein is as set forth herein, with the proviso that the encoded amino acid sequence is not the wild-type or parental sequence or a naturally occurring amino acid sequence and/or that the nucleotide sequence is not a wild-type or naturally occurring nucleotide sequence. A naturally occurring amino acid or nucleotide sequence is understood by those skilled in the art as relating to a sequence that is found in a naturally occurring organism as found in nature. Thus, a nucleotide or amino acid sequence that is not found in the same state or having the same nucleotide or encoded amino acid sequence as in a naturally occurring organism is included within the meaning of a recombinant nucleotide and/or amino acid sequence described herein. For example, a nucleotide or amino acid sequence that has been altered at one or more nucleotide or amino acid positions from a parent sequence, including variants as described herein, are included within the meaning of a nucleotide or amino acid sequence described herein that is not naturally occurring. A recombinant nucleic acid described herein excludes a naturally occurring chromosome that contains the nucleotide sequence, and can further exclude other molecules, as found in a naturally occurring cell, such as DNA binding proteins, for example, proteins such as histones that bind to chromosomes within a eukaryotic cell.

[00187] Thus, a recombinant nucleic acid described here has physical and chemical differences compared to a naturally occurring nucleic acid. A recombinant or non-naturally occurring nucleic acid described herein does not contain or does not necessarily have some or all of the chemical bonds, either covalent or non-covalent bonds, of a naturally occurring nucleic acid as found in nature. A recombinant nucleic acid described herein thus differs from a naturally occurring nucleic acid, for example, by having a different chemical structure than a naturally occurring nucleic acid as found in a chromosome. A different chemical structure can occur, for example, by cleavage of phosphodiester bonds that release a recombinant nucleic acid from a naturally occurring chromosome. A recombinant nucleic acid described herein can also differ from a naturally occurring nucleic acid by isolating or separating the nucleic acid from proteins that bind to chromosomal DNA in either prokaryotic or eukaryotic cells, thereby differing from a naturally occurring nucleic acid by different non-covalent bonds. With respect to nucleic acids of prokaryotic origin, a non- naturally occurring nucleic acid described herein does not necessarily have some or all of the naturally occurring chemical bonds of a chromosome, for example, binding to DNA binding proteins such as polymerases or chromosome structural proteins, or is not in a higher order structure such as being supercoiled. With respect to nucleic acids of eukaryotic origin, a non- naturally occurring nucleic acid described herein also does not contain the same internal nucleic acid chemical bonds or chemical bonds with structural proteins as found in chromatin. For example, a non-naturally occurring nucleic acid described herein is not chemically bonded to histones or scaffold proteins and is not contained in a centromere or telomere. Thus, the non-naturally occurring nucleic acids described herein are chemically distinct from a naturally occurring nucleic acid because they either lack or contain different van der Waals interactions, hydrogen bonds, ionic or electrostatic bonds, and/or covalent bonds from a nucleic acid as found in nature. Such differences in bonds can occur either internally within separate regions of the nucleic acid (that is cis) or such difference in bonds can occur in trans, for example, interactions with chromosomal proteins. In the case of a nucleic acid of eukaryotic origin, a cDNA is considered to be a recombinant or non-naturally occurring nucleic acid since the chemical bonds within a cDNA differ from the covalent bonds, that is the sequence, of a gene on chromosomal DNA. Thus, it is understood by those skilled in the art that recombinant or non-naturally occurring nucleic acid is distinct from a naturally occurring nucleic acid.

[00188] In some embodiments, provided herein is a recombinant nucleic acid that includes a nucleotide sequence encoding an engineered lasso peptide described herein or a lasso precursor peptide described herein that is operatively linked to a promoter. Such a promoter can facilitate expression of the engineered lasso peptide or lasso precursor peptide in a microbial organism as described herein.

[00189] In some embodiments, provided herein is a vector containing a recombinant nucleic acid described herein. In some embodiments, the vector is an expression vector. In some embodiments, the vector comprises double stranded DNA.

7.4.3 Biosynthesis of Engineered Lasso Peptides

[00190] In a related aspect of the present disclosure, provided herein are methods and systems for producing engineered lasso peptides. In some embodiments, the methods provided herein can produce a large quantities of matured, functional lasso peptides in a short period of time.

7.4.3.1 Natural Lasso Peptides and Genomic Mining Tools

[00191] Some naturally existing lasso peptides are encoded by a lasso peptide biosynthetic gene cluster, which typically comprises three main genes: one encodes for a lasso precursor peptide (referred to as Gene A), and two encode for processing enzymes including a lasso peptidase (referred to as Gene B) and a lasso cyclase (referred to as Gene C). The lasso precursor peptide comprises an engineered lasso core peptide and additional peptidic fragments known as the leader sequence that facilitates recognition and processing by the processing enzymes. The leader sequence can determine substrate specificity of the processing enzymes. The processing enzymes encoded by the lasso peptide gene cluster convert the lasso precursor peptide into a matured lasso peptide having the lariat-like topology. Particularly, the lasso peptidase removes additional sequences from the precursor peptide to generate an engineered lasso core peptide, and the lasso cyclase cyclizes the N- terminal portion of the core peptide around the C-terminal tail portion to form the lariat-like topology. Some lasso gene clusters further encodes for additional protein elements that facilitates the lasso formation and/or post-translational modification, including a facilitator protein known as the post-translationally modified peptide (RiPP) recognition element (RRE). Some lasso gene clusters further encodes for lasso peptide transporters, kinases, or proteins that play a role in immunity, such as isopeptidase (Burkhart, B.J., et al., Nat. Chem. Biol., 2015, 11, 564-570; Knappe, T.A. et al., J. Am. Chem. Soc., 2008, 130, 11446-11454; Solbiati, J.O. et al. J. Bacteriol., 1999, 181, 2659-2662; Fage, C.D., et al., Angew. Chem. Int. Ed., 2016, 55, 12717 -12721; Zhu, S., et al., J. Biol. Chem. 2016, 291, 13662-13678). [00192] Computer-based genome-mining tools can be used to identify lasso biosynthetic gene clusters based on known genomic information. For example, one algorithm known as RODEO can rapidly analyze a large number of biosynthetic gene clusters (BGCs) by predicting the function for genes flanking query proteins. This is accomplished by retrieving sequences from a genome sequence database, such as GenBank, followed by analysis with, for example, the hidden Markov model alignment tool HMMER3. The results are compared against the Pfam database with the data being returned to the users in the form of spreadsheet. For analysis of BGCs not covered by Pfam, RODEO allows usage of additional pHMMs (either curated databases or user-generated). Taking advantage of RODEO’s ability to rapidly analyze genes neighboring a query, it is possible to compile a list of all observable lasso peptide biosynthetic gene clusters in GeneBank (Online Methods). A comprehensive evaluation of this data set would provide great insight into the lasso peptide family. Lasso peptide biosynthetic gene clusters can be identified by looking for the local presence of genes encoding proteins matching the Pfams for the lasso cyclase, lasso peptidase, and RRE. [00193] To confidently predict lasso precursors, RODEO next performed a six-frame translation of the intergenic regions within each of the identified potential lasso biosynthetic gene clusters. The resulting peptides can be assessed based on length and essential sequence features and split into predicted leader and core regions. A series of heuristics based on known lasso peptide characteristics can be defined to predict precursors from a pool of false positives. After optimization of heuristic scoring, good prediction accuracy for biosynthetic gene clusters closely related to known lasso peptides can be obtained.

[00194] Machine learning, particularly, support vector machine (SVM) classification, would be effective in locating precursor peptides from predicted BGCs more distant to known lasso peptides. SVM is well-suited for RiPP discovery due to availability of SVM libraries that perform well with large data sets with numerous variables and the ability of SVM to minimize unimportant features. The SVM classifier can be optimized using a randomly selected and manually curated training set from the unrefined whole data. Of these, a random subpopulation was withheld as a test set to avoid over-fitting. By combining SVM classification with motif (MEME) analysis, along with the original heuristic scoring, prediction accuracy can be greatly enhanced as evaluated by recall and precision metrics. This tripartite procedure can yield a high-scoring, well-separated population of lasso precursor peptide from candidate peptides. The training set can be found to display nearly identical scoring distributions upon comparison to the full data set.

[00195] Other examples of genomic or biosynthetic gene search engine that can be used in connection with the present disclosure include the WARP DRIVE BIO™ software, anti- SMASH (ANTI-SMASH™) software (See: BHn, K ., et al.. Nucleic Acids Res., 2017, 45, W36-W41), iSNAP™ algorithm (See: Ibrahim, A., et al., Proc. Nat. Acad. Sci., USA., 2012, 109, 19196-19201), CLUSTSCAN™ (Starcevic, et al., Nucleic Acids Res., 2008, 36, 6882- 6892), NP searcher (Li et al. (2009) Automated genome mining for natural products. BMC Bioinformatics, 10, 185), SBSPKS™ (Anand, et al. Nucleic Acids Res. , 2010, 38, W487- W496), BAGEL3™ (Van Heel, et al., Nucleic Acids Res., 2013, 41, W448-W453), SMURF™ (Khaldi et al., Fungal Genet. Biol., 2010, 47, 736-741), ClusterFinder (CLUSTERFINDER™) or ClusterBlast (CLUSTERBLAST™) algorithms, and an Integrated Microbial Genomes (IMG)-ABC system (DOE loint Genome Institute (JGI)). In some embodiments, lasso peptide biosynthetic gene clusters for use in CFB methods and processes as provided herein are identified by mining genome sequences of known bacterial natural product producers using established genome mining tools, such as anti-SMASH, BAGEL3, and RODEO. These genome mining tools can also be used to identify novel biosynthetic genes (e.g., for use in CFB systems and processes as provided herein) within metagenomic based DNA sequences. Lasso peptide biosynthetic gene clusters can be used in the methods and systems described herein to produce various lasso peptides.

7.4.3.1 Cell-free Biosynthesis of Engineered Lasso Peptides

[00196] In one aspect, provided herein are methods for producing engineered lasso peptides as described herein (e.g., Table 1) in vitro cell-free biosynthesis (CFB) methods. CFB methods employ the enzymes and the biosynthetic and metabolic machinery present inside cells, but without using living cells. CFB methods allow rapid expression of natural biosynthetic genes and pathways and facilitate targeted or phenotypic activity screening of natural products, without the need for plasmid-based cloning or in vivo cellular propagation, thus enabling rapid process/product pipelines (e.g., creation of large number of lasso peptides in a short time). Features of the CFB methods for lasso peptide production include that oligonucleotides (linear or circular constructs of DNA or RNA) encoding a minimal set of lasso peptide biosynthesis pathway genes (e.g., Genes A-C in a lasso peptide biosynthetic gene cluster) can be added to a cell extract containing in vitro transcription-translation (TX- TL) machinery for transcribing and translating the genes into the functional enzymes and lasso precursor peptides for production of lasso peptides. Accordingly, the CFB methods can produce in a CFB reaction mixture at least two or more of the lasso peptide variants as described in Si, et Am. Chem. Soc., 143:5917-5927 (2021).

[00197] In some embodiments, the method for producing an engineered lasso peptide comprises (a) providing a CFB system comprising a minimal set of lasso peptide biosynthesis components; and (b) incubating the CFB system under a suitable condition to produce the engineered lasso peptide.

[00198] In some embodiments, the minimal set of lasso peptide biosynthesis components comprises one or more components functions to provide a lasso precursor peptide, and one or more components function to process the lasso precursor peptide into the engineered lasso peptide. In some embodiments, the one or more components function to process the lasso precursor peptide into the engineered lasso peptide consist of a lasso peptidase and a lasso cyclase. In some embodiments, the one or more components function to process the lasso precursor peptide into the engineered lasso peptide consists of a lasso peptidase, a lasso cyclase and an RRE. [00199] In some embodiments, the minimal set of lasso peptide biosynthesis components comprises one or more components functions to provide an engineered lasso core peptide, and one or more components function to process the lasso core peptide into the engineered lasso peptide. In some embodiments, the one or more components function to process the lasso core peptide into the engineered lasso peptide comprises one or more selected from a lasso peptidase, a lasso cyclase and an RRE. In some embodiments, the one or more components function to process the lasso core into the engineered lasso peptide consist of a lasso cyclase.

[00200] In various embodiments, the one or more components function to provide a peptide or protein (e.g., a lasso precursor peptide, an engineered lasso core peptide, or lasso peptide biosynthetic enzymes and proteins) in a CFB system can be provided in the form of the peptide or protein are provided in the form of the peptide or protein per se.

[00201] In some embodiments, at least some of the peptide or protein components in the CFB system can be natural peptides or polypeptides. In some embodiments, at least some of the peptide or protein components in the CFB system are derivatives of natural peptides or polypeptides. In some embodiments, at least some of the peptide or protein components in the CFB system are non-natural peptides. In some embodiments, the one or more peptide or protein components of the CFB system can be isolated from nature, such as isolated from microorganisms producing the lasso precursor peptides. In some embodiments, the one or more peptide or protein components of the CFB system can be synthetically or recombinantly produced, using methods known in the art. In some embodiments, the one or more peptide or protein components of the CFB system can be synthesized using the CFB system as described herein, followed by purifying the biosynthesized peptide or protein components from the CFB system.

[00202] In some embodiments, the CFB system comprises one or more fusion protein, or a polynucleotide encoding the fusion protein such that the CFB system is capable of producing the fusion protein through in vitro TX-TL.

[00203] In some embodiments, the fusion protein comprised a lasso precursor peptide or an engineered lasso core peptide fused to one or more lasso peptide biosynthesis components. In some embodiments, the one or more lasso peptide biosynthesis components are selected from (i) a lasso peptidase; (ii) a lasso cyclase; (iii) a RRE; or (iv) any combinations of (i) to (iii). In some embodiments, the one or more lasso peptide biosynthesis components are encoded by the same lasso peptide biosynthetic gene cluster. In other embodiments, the one or more lasso peptide biosynthesis components are encoded by different lasso peptide biosynthetic gene cluster.

[00204] In some embodiments, the fusion protein comprises an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not comprise an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide. [00205] In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso cyclase. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a RRE. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase and a lasso cyclase. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase and a RRE. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso cyclase and a RRE. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase, a lasso cyclase and RRE. In specific embodiments, the fusion protein comprises an engineered lasso core peptide fused to a lasso peptidase. In specific embodiments, the fusion protein comprises an engineered lasso core peptide fused to a lasso cyclase. In specific embodiments, the fusion protein comprises an engineered lasso core peptide fused to a RRE. In specific embodiments, the fusion protein comprises an engineered lasso core peptide fused to a lasso peptidase and a lasso cyclase. In specific embodiments, the fusion protein comprises an engineered lasso core peptide fused to a lasso peptidase and a RRE. In specific embodiments, the fusion protein comprises an engineered lasso core peptide fused to a lasso cyclase and a RRE. In specific embodiments, the fusion protein comprises an engineered lasso core peptide fused to a lasso peptidase, a lasso cyclase and RRE.

[00206] In some embodiments, the fusion protein comprised a lasso precursor peptide or an engineered lasso core peptide fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide comprises a peptide or polypeptide that facilitates production of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom through cell-free biosynthesis. Examples of peptide or polypeptide that can be fused with a lasso precursor peptide or an engineered lasso core peptide according to the present disclosure include but are not limited to (i) a peptide or polypeptide that increases the level of transcription of the lasso precursor peptide or lasso core peptide in the CFB system; (ii) a peptide or polypeptide that increases the level of translation of the lasso precursor peptide or lasso core peptide in the CFB system; (iii) a peptide or polypeptide that facilitates the processing of the lasso precursor peptide or lasso core peptide into the engineered lasso peptide; (iv) a peptide or polypeptide that improves stability of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; (v) a peptide or polypeptide that improves solubility of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; (vi) a peptide or polypeptide that enables or facilitates the detection of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; (vii) a peptide or polypeptide that enables or facilitates purification of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; (viii) a peptide or polypeptide that enables or facilitates immobilization of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; or (ix) any combination of (i) to (viii).

[00207] In some embodiments, the fusion protein comprised a lasso precursor peptide or an engineered lasso core peptide fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide comprises a biologically active peptide or polypeptide. Examples of biologically active peptide or polypeptide that can be fused with a lasso precursor peptide or lasso core peptide according to the present disclosure include but are not limited to (i) a peptide or polypeptide capable of binding to a target molecule (e.g., an antibody or an antigen); (ii) a peptide or polypeptide that enhance cell permeability of the fusion protein; (iii) a peptide or polypeptide capable of conjugating the fusion protein to at least one additional copy of the fusion protein; (iv) a peptide or polypeptide capable of linking the fusion protein to one or more peptidic or non- peptidic molecule; (v) a peptide or polypeptide capable of modulating activity of the lasso precursor peptide or lasso core peptide; (vi) a peptide or polypeptide capable of modulating activity of the lasso peptide derived from the lasso precursor peptide or the lasso core peptide; or (vii) any combinations of (i) to (vi).

[00208] In some embodiments, the fusion protein comprised a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide is fused to the N-terminus of the lasso peptidase or the lasso cyclase. In some embodiments, the one or more additional peptide or polypeptide is fused at the C-terminus of the lasso peptidase or the lasso cyclase. In some embodiments, a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the lasso peptidase or the lasso cyclase, wherein the 5’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide. In some embodiments, a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the lasso peptidase or the lasso cyclase, wherein the 3’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide. In some embodiments, the fusion protein comprises an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not comprise an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide.

[00209] In some embodiments, the fusion protein comprised a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide. In some embodiments, the more additional peptide or polypeptide comprises a peptide or polypeptide encoded by a lasso peptide biosynthetic gene cluster. Examples of peptide or polypeptide that can be fused with a lasso precursor peptide or an engineered lasso core peptide according to the present disclosure include but are not limited to (i) a lasso precursor peptide; (ii) an engineered lasso core peptide; (iii) a lasso peptidase; (iv) a lasso cyclase, (v) a RRE; or (vi) any combinations of (i) to (vi). In specific embodiments, the fusion protein comprises at least one lasso cyclase and at least one lasso peptidase. In specific embodiments, the fusion protein comprises at least one lasso cyclase fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso peptidase fused to a RRE.

[00210] In some embodiments, the fusion protein comprised a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide comprises a peptide or polypeptide that facilitates production of the lasso peptidase or lasso cyclase through cell-free biosynthesis. Examples of peptide or polypeptide that can be fused with the lasso peptidase or lasso cyclase according to the present disclosure include but are not limited to (i) a peptide or polypeptide that increases the level of transcription of the lasso peptidase or lasso cyclase in the CFB system; (ii) a peptide or polypeptide that increases the level of translation of the lasso peptidase or lasso cyclase in the CFB system; (iii) a peptide or polypeptide that improves stability of the lasso peptidase or lasso cyclase; (vi) a peptide or polypeptide that improves solubility of the lasso peptidase or lasso cyclase; (v) a peptide or polypeptide that enables or facilitates the detection of the lasso peptidase or lasso cyclase; (vi) a peptide or polypeptide that enables or facilitates purification of the lasso peptidase or lasso cyclase; (vii) a peptide or polypeptide that enables or facilitates immobilization of the lasso peptidase or lasso cyclase; or (viii) any combination of (i) to (vii).

[00211] In some embodiments, the fusion protein comprised a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide comprises a biologically active peptide or polypeptide. Examples of biologically active peptide or polypeptide that can be fused with a lasso peptidase or a lasso cyclase according to the present disclosure include but are not limited to (i) a peptide or polypeptide capable of modulating the reaction catalyzing activity of the lasso peptidase or lasso cyclase; (ii) a peptide or polypeptide capable of modulating target specificity of the lasso peptidase or lasso cyclase; (iii) an enzyme having the same or different enzymatic activity as the lasso peptidase or lasso cyclase; or any combination of (i) to (iii).

[00212] In some embodiments, the fusion protein comprised a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide is fused to the N-terminus of the RRE. In some embodiments, the one or more additional peptide or polypeptide is fused at the C-terminus of the RRE. In some embodiments, a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the RRE, wherein the 5’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide. In some embodiments, a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the RRE, wherein the 3’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide. In some embodiments, the fusion protein comprises an amino acid linker between the RRE and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not comprise an amino acid linker between RRE and the one or more additional peptide or polypeptide.

[00213] In some embodiments, the fusion protein comprised a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide. In some embodiments, the more additional peptide or polypeptide comprises a peptide or polypeptide encoded by a lasso peptide biosynthetic gene cluster. Examples of peptide or polypeptide that can be fused with a lasso precursor peptide or an engineered lasso core peptide according to the present disclosure include but are not limited to (i) a lasso precursor peptide; (ii) an engineered lasso core peptide; (iii) a lasso peptidase; (iv) a lasso cyclase, (v) a RRE; or (vi) any combinations of (i) to (vi). In specific embodiments, the fusion protein comprises at least one lasso precursor peptide fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso core peptide fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso cyclase fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso peptidase fused to a RRE.

[00214] In some embodiments, the fusion protein comprised a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide comprises a peptide or polypeptide that facilitates production of the RRE through cell-free biosynthesis. Examples of peptide or polypeptide that can be fused with the RRE according to the present disclosure include but are not limited to (i) a peptide or polypeptide that increases the level of transcription of the RRE in the CFB system; (ii) a peptide or polypeptide that increases the level of translation of the RRE in the CFB system; (iii) a peptide or polypeptide that improves stability of the RRE; (vi) a peptide or polypeptide that improves solubility of the RRE; (v) a peptide or polypeptide that enables or facilitates the detection of the RRE; (vi) a peptide or polypeptide that enables or facilitates purification of the RRE; (vii) a peptide or polypeptide that enables or facilitates immobilization of the RRE; or (viii) any combination of (i) to (vii).

[00215] In some embodiments, the fusion protein comprised a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide comprises a biologically active peptide or polypeptide. Examples of biologically active peptide or polypeptide that can be fused with a RRE according to the present disclosure include but are not limited to (i) a peptide or polypeptide capable of modulating the reaction catalyzing activity of the lasso peptidase or lasso cyclase; (ii) a peptide or polypeptide capable of modulating target specificity of the lasso peptidase or lasso cyclase; (iii) an enzyme having the same or different enzymatic activity as the lasso peptidase or lasso cyclase; or any combination of (i) to (iii).

[00216] In particular embodiments, the lasso precursor peptide genes are fused at the 5’- terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, such as sequences encoding maltose-binding protein (MBP) or small ubiquitin-like modifier protein (SUMO), which enhance the stability, solubility, and production of the desired TX-TL products (Marblestone, J.G., el ct!.. Protein Sci, 2006, 15, 182-189). In particular embodiments, the lasso precursor peptides are fused at the C- terminus of the leader sequences to form conjugates with peptides or proteins, such as maltose-binding protein or small ubiquitin-like modifier protein, which enhance the stability, solubility, and production of the fused MBP-lasso or SUMO-lasso precursor peptide.

[00217] In particular embodiments, the lasso precursor peptide genes or lasso core peptide genes are fused at the 3 ’-terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, such as sequences encoding maltose-binding protein (MBP) or small ubiquitin-like modifier protein (SUMO), which enhance the stability, solubility, and production of the desired TX-TL products. In particular embodiments, the lasso precursor peptides, lasso core peptides, or lasso peptides are fused at the N-terminus to form conjugates with peptides or proteins, such as maltose-binding protein or small ubiquitin- like modifier protein, which enhance the stability, solubility, and production of the fused MBP-lasso or SUMO-lasso precursor peptide.

[00218] In particular embodiments, the lasso precursor peptide genes or lasso core peptide genes are fused at the 5'-terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, with or without a linker, such as sequences encoding peptide tags for affinity purification or immobilization, including his-tags, strep- tags, or FLAG-tags. In some embodiments, the lasso precursor peptides, lasso core peptides, or lasso peptides are fused at the C-terminus of the core peptides to form conjugates with other peptides or proteins, with or without a linker, such as peptide tags for affinity purification or immobilization, including his-tags, strep-tags, or FLAG-tags.

[00219] In particular embodiments, lasso precursor peptides, lasso core peptides, or lasso peptides are fused to molecules that can enhance cell permeability or penetration into cells, for example through the use of arginine-rich cell-penetrating peptides such as TAT peptide, penetratin, and flock house virus (FHV) coat peptide (Brock, R., Bioconjug. Chem., 2014, 25, 863-868). In particular embodiments, a lasso precursor peptide gene or core peptide gene is fused at the 3 ’-terminus to oligonucleotide sequences that encode arginine-rich cell- penetrating peptides or proteins, including oligonucleotide sequences that encode penetratin, and flock house virus (FHV) coat peptide or similar peptides that contain guanidinium groups or a combination of lysine and guanidinium groups (Wender, P.A., el al., Adv. Drug Deliv. Rev., 2008, 60, 452-472). In particular embodiments, a lasso precursor peptide, lasso core peptide, or lasso peptide is fused at the C-terminus to peptides that promote cell penetration such as arginine-rich cell-penetrating peptides or proteins, including amino acid sequences that encode TAT peptide, penetratin, and flock house virus (FHV) coat peptide or similar peptides that contain guanidinium groups or a combination of lysine and guanidinium groups. [00220] In particular embodiments, the lasso precursor peptide genes or lasso core peptide genes are fused at the 5'-terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, with or without a linker, such as sequences encoding peptide epitopes that are known to bind with high affinity to antibodies, cell surface proteins, or cell surface receptors, including cytokine binding epitopes, integrin ligand binding epitopes, and the like. In particular embodiments, the lasso precursor peptides, lasso core peptides, or lasso peptides are fused at the C-terminus to peptides or proteins, with or without a linker, such as peptide epitopes that are known to bind with high affinity to antibodies, cell surface proteins, or cell surface receptors, including cytokine binding epitopes, integrin ligand binding epitopes, and the like.

[00221] Additionally or alternatively, the one or more components function to provide a peptide or protein (e.g., a lasso precursor peptide, an engineered lasso core peptide, or lasso peptide biosynthetic enzymes and proteins) in a CFB system can be provided in the form of a nucleic acid encoding the peptide or protein and in vitro TX-TL machinery capable of producing the peptide or protein vial in vitro TX-TL of the coding sequences. In various embodiments, the coding nucleic acid can be DNA, RNA or cDNA. In various embodiments, one or more coding nucleic acid sequences can be contained in the same nucleic acid molecule, such as a vector.

[00222] It is understood that when more than one coding nucleic acid sequences are included in a CFB system, such more than one encoding nucleic acid sequences can be introduced on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof. For example, as disclosed herein, a microbial organism or a cell extract can be engineered to express two or more exogenous nucleic acids encoding lasso precursor peptide, lasso core peptide, lasso peptidase, lasso cyclase or RRE. In the case where two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism or into a cell extract, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid or as linear strands of DNA, or on separate plasmids, or can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids can be introduced into a host organism or into a cell extract in any desired combination, for example, on a single plasmid, or on separate plasmids, or as linear strands of DNA, or can be integrated into the host chromosome at a single site or multiple sites.

[00223] In some embodiments, the in vitro TX-TL machinery is purified from a host cell. In some embodiments, the in vitro TX-TL machinery is provided in the form of a cell extract of a host cell. An exemplary procedure for obtaining a cell extract comprises the steps of (i) growing cells, (ii) breaking open or lysing the cells by mechanical, biological or chemical means, (iii) removing cell debris and insoluble materials e.g., by filtration or centrifugation, and (iv) optionally treating to remove residual RNA and DNA, but retaining the active enzymes and biosynthetic machinery for transcription and translation, and optionally the metabolic pathways for co-factor recycle, including but not limited to co-factors such as THF, S-adenosylmethionine, ATP, NADH, NAD and NADP and NADPH. In some embodiments, a cell extract can be further supplemented for improved performance in in vitro TX-TL.

[00224] In some embodiments, a cell extract can be further supplemented with some or all of the twenty proteinogenic naturally-occurring amino acids and corresponding transfer ribonucleic acids (tRNAs), and optionally, can be supplemented with additional components, including but not limited to: (1) glucose, xylose, fructose, sucrose, maltose, or starch, (2) adenosine triphosphate (ATP), and/or adenosine diphosphate (ADP), purine and guanidine nucleotides, adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, and/or uridine triphosphate, or combinations thereof, (3) cyclic-adenosine monophosphate (cAMP) and/or 3 -phosphoglyceric acid (3-PGA), (4) nicotinamide adenine dinucleotides NADH and/or NAD, or nicotinamide adenine dinucleotide phosphates, NADPH, and/or NADP, or combinations thereof, (5) amino acid salts such as magnesium glutamate and/or potassium glutamate, (6) buffering agents such as HEPES, TRIS, spermidine, or phosphate salts, (7) inorganic salts, including but not limited to, potassium phosphate, sodium chloride, magnesium phosphate, and magnesium sulfate, (8) cofactors such as folinic acid and co- enzyme A (CoA), L(-)-5-formyl-5,6,7,8-tetrahydrofolic acid (THF), and/or biotin, (8) RNA polymerase, (9) 1,4-dithiothreitol (DTT), (10) magnesium acetate, and/or ammonium acetate, and/or (11) crowding agents such as PEG 8000, Ficoll 70, or Ficoll 400, or combinations thereof. In some embodiments, the cell extracts or supplemented cell extracts can be used as a reaction mixture to carry out in vitro TX-TL. In some embodiments, supplementations or adjustments can be made to the cell extract to provide a suitable condition for lasso formation. [00225] In some embodiments, the in vitro TX-TL machinery is provided in the form of a cell extract or supplemented cell extract of a host cell. In some embodiments, the host cell is the cell of the same organism where the coding nucleic acid is derived from. For CFB of lasso peptides and related molecules thereof, the coding nucleic acid sequences can be identified using one or more computer-based genomic mining tools described herein or known in the art. For example, U.S. Provisional Application Nos. 62/652,213 and 62/651,028 disclose thousands of sequences from lasso peptide biosynthetic gene clusters identified from various organisms, and provide GenBank accession numbers for various sequences for lasso precursor peptides, lasso peptidase, lasso cyclase and/or RRE. Host organisms where the lasso peptide biosynthetic gene clusters originate can be identified based on the GenBank accession numbers, including but not limited to Caulobacteraceae species (e.g., Caulobacter sp. K31, Caulobacter henricii). Streptomyces species (e.g. Streptomyces nodosus. Streptomyces caatingaensis . Burkholderiaceae species (e.g., Burkholderia thailandensis E264), Pseudomallei species, Bacillus species, Burkholderia species (e.g., Burkholderia thailandensis MSMB43, Burkholderia oklahomensis, Burkholderia pseudomallei), Sphingomonadaceae species (e.g., Sphingobium sp. YBL2, Sphingobium chlorophenolicum, Sphingobium yanoikuyae). In other embodiments, the host cell is a microbial organism known to be applicable to fermentation processes. Exemplary bacteria include species selected from Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Streptomyces albus, Clostridium acetobutylicum, Vibrio natriegens, Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts or fungi include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger and Pichia pastoris. E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering. Other particularly useful host organisms include Vibrio natriegens, and yeast such as Saccharomyces cerevisiae.

[00226] In some embodiments, the CFB system is configured to produce an engineered lasso peptide. In specific embodiments, the CFB system comprises one or more components configured to provide (i) a lasso precursor peptide, (ii) a lasso peptidase, (iii) a lasso cyclase. In specific embodiments, the CFB system comprises one or more components configured to provide (i) an engineered lasso core peptide, and (ii) a lasso cyclase. In some embodiments, the CFB system further comprises one or more components configured to provide (iv) an RRE. In some embodiments, all of (i) to (iv) above are provided in the CFB system as the corresponding peptide or protein. In alternative embodiments, at least one of (i) to (iv) above is provided in the CFB system as a nucleic acid encoding the corresponding protein, and the CFB system further comprises in vitro TX-TL machinery for producing the corresponding protein from the coding nucleic acid. In these embodiments, the CFB systems can be incubated under a condition suitable for lasso formation to produce the engineered lasso peptide. The incubation condition can be designed and adjusted based on various factors known to skilled artisan in the art, including for example, condition suitable for maintain stability of components of the CFB system, conditions suitable for the lasso processing enzymes to exert enzymatic activities, and/or conditions suitable for the in vitro TX-TL of the coding sequences present in the CFB system.

[00227] Without being bound by the theory, it is contemplated that different lasso peptidase can process the same lasso precursor peptide into different lasso core peptide by recognizing and cleaving different leader peptide off the lasso precursor. Additionally, different lasso cyclase can process the same lasso core peptide into distinct lasso peptides by cyclizing the core peptide at different ring-forming amino acid residues. Additionally, different RREs can facilitate different processing by the lasso peptidase and/or lasso cyclase, and thus lead to formation of distinct lasso peptides from the same lasso precursor peptide. [00228] Accordingly, in some embodiments, to produce a natural lasso peptide, the CFB system comprises the lasso precursor peptide, lasso peptidase, and lasso cyclase produced from coding sequences of the same lasso peptide biosynthetic gene cluster (such as Genes A, B, and C of the same lasso peptide biosynthetic gene cluster). In some embodiments, to produce a natural lasso peptide, the CFB system comprises the lasso precursor peptide, lasso peptidase, lasso cyclase, and RRE produced from coding sequences of the same lasso peptide biosynthetic gene cluster.

[00229] In some embodiments, to produce a natural lasso peptide, the CFB system comprises the lasso core peptide, and lasso cyclase produced from coding sequences of the same lasso peptide biosynthetic gene cluster (such as Genes A and C of the same lasso peptide biosynthetic gene cluster). In some embodiments, to produce a natural lasso peptide, the CFB system comprises the lasso core peptide, lasso cyclase, and RRE produced from coding sequences of the same lasso peptide biosynthetic gene cluster. [00230] In alternative embodiments, to produce a derivative of a natural lasso peptide, at least two of the lasso precursor peptide, lasso peptidase and lasso cyclase in the CFB system are produced from coding sequences of different lasso peptide biosynthetic gene clusters (such as Gene A from one, and Genes B and C from another, lasso peptide biosynthetic gene cluster). In alternative embodiments, to produce a derivative of a natural lasso peptide, at least two of the lasso precursor peptide, lasso peptidase, lasso cyclase and RRE in the CFB system are produced from coding sequences of different lasso peptide biosynthetic gene clusters.

[00231] In alternative embodiments, to produce a derivative of a natural lasso peptide, the lasso core peptide and lasso cyclase in the CFB system are produced from coding sequences of different lasso peptide biosynthetic gene clusters (such as Gene A from one, and Gene C from another, lasso peptide biosynthetic gene cluster). In alternative embodiments, to produce a derivative of a natural lasso peptide, at least two of the lasso core peptide, lasso cyclase and RRE in the CFB system are produced from coding sequences of different lasso peptide biosynthetic gene clusters.

[00232] In some embodiments, to produce a derivative of a natural lasso peptide, a lasso precursor peptide is modified at the core peptide sequence, while the leader sequence is maintained the same. The modified precursor peptide can then processed by corresponding lasso peptidase and/or lasso cyclase into a matured engineered lasso peptide with modified amino acid sequence. For example, in specific embodiments, a lasso precursor peptide has an amino acid sequence as described herein (e.g., Table 1) and a leader sequence comprising the amino acid sequence of MTEQSEQTPTEYIPPMLVEVGEFTEDTL (SEQ ID NO: 118). This leader sequence is recognized by lasso peptide biosynthesis component proteins, and hence such precursor peptides can be processed by the same lasso peptide biosynthesis component proteins into matured engineered lasso peptides having different amino acid sequences as described herein.

[00233] Accordingly, in specific embodiments, the present method of cell-free biosynthesis of an engineered lasso peptide comprises (a) contacting a lasso precursor peptide described herein with a lasso peptide biosynthesis component in a cell-free biosynthesis reaction mixture; and (b) incubating the cell-free biosynthesis reaction mixture under a condition suitable for lasso formation to produce the engineered lasso peptide. In some embodiments, the present method of cell-free biosynthesis of an engineered lasso peptide comprises (a) contacting a lasso precursor peptide with a lasso peptide biosynthesis component comprising a lasso cyclase in a cell-free biosynthesis reaction mixture; and (b) incubating the cell-free biosynthesis reaction mixture under a condition suitable for lasso formation to produce the engineered lasso peptide. In some embodiments, the lasso peptide biosynthesis component further comprises a lasso peptidase and/or a RRE. In some embodiments, the present method of cell-free biosynthesis of an engineered lasso peptide comprises (a) contacting a lasso precursor peptide having an amino acid sequence as described herein (e.g., Table 1) and a leader sequence comprising the amino acid sequence of MTEQSEQTPTEYIPPMLVEVGEFTEDTL (SEQ ID NO: 118) with a lasso peptide biosynthesis component comprising a lasso peptidase and lasso cyclase in a cell-free biosynthesis reaction mixture; and (b) incubating the cell-free biosynthesis reaction mixture under a condition suitable for lasso formation to produce the engineered lasso peptide. In some embodiments, the lasso peptide biosynthesis component further comprises a RRE. [00234] In various embodiments, the contacting step (a) comprises adding a first nucleic acid sequence encoding the peptide into the cell-free biosynthesis reaction mixture, and where the cell-free biosynthesis reaction mixture comprises in vitro TX-TL machinery and is configured to express the peptide. In some embodiments, the contacting step (a) comprises adding a second nucleic acid sequence encoding the lasso peptide biosynthesis component to the cell-free biosynthesis reaction mixture, and where the cell-free biosynthesis reaction mixture comprises in vitro TX-TL machinery configured to express the lasso peptide biosynthesis component. In some embodiments, the lasso peptide biosynthesis component comprises a lasso peptidase. In some embodiments, the lasso peptide biosynthesis component comprises a lasso cyclase. In some embodiments, the lasso peptide biosynthesis component further comprises a post-translationally modified peptide (RiPP) recognition element (RRE). [00235] More particularly, in some of those embodiments where the lasso peptide biosynthesis component comprises a lasso peptidase and a lasso cyclase, the contacting step (a) comprises adding the second nucleic acid sequence encoding the lasso cyclase and a third nucleic acid sequence encoding the lasso peptidase. In some of those embodiments where the lasso peptide biosynthesis component comprises a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE), the contacting step (a) comprises adding the second nucleic acid sequence encoding the lasso cyclase and a fourth nucleic acid sequence encoding the RRE. In some of those embodiments where the lasso peptide biosynthesis component comprises a lasso peptidase, a lasso cyclase and a post- translationally modified peptide (RiPP) recognition element (RRE), and where the contacting step (a) comprises adding the second nucleic acid sequence encoding the lasso cyclase, a third nucleic acid sequence encoding the lasso peptidase and a fourth nucleic acid sequence encoding the RRE. In some embodiments, at least two of the first, second, third and fourth nucleic acid sequences are in a same nucleic acid molecule. In some embodiments, the engineered lasso peptide comprises an amino acid sequence as described herein (e.g., Table 1) and is G1-D9 cyclized. In some embodiments, the cell-free biosynthesis reaction mixture comprises cell extract or supplemented cell extract.

[00236] Additional lasso peptide biosynthesis components and corresponding leader sequences are known in the art, such as those disclosed in PCT application publication numbers: WO2019/191571, which is incorporated herein by reference in its entirety. Therefore, in some embodiments, to produce engineered lasso peptides having amino acid sequences as described herein (e.g., Table 1), the core peptide sequences can be fused to any known leader sequence, thereby producing a lasso precursor peptide, and the method then employs one or more lasso peptide biosynthesis component capable of recognizing such leader sequence and processing the lasso precursor peptide into matured engineered lasso peptides having an amino acid sequence as described herein (e.g., Table 1).

[00237] Accordingly, in some embodiments, provided herein is a method of cell-free biosynthesis of an engineered lasso peptide having an amino acid sequence as described herein (e.g., Table 1), wherein the method comprises (a) contacting a lasso precursor peptide comprising a leader sequence and an engineered lasso core peptide sequence as described herein (e.g, Table 1) with a lasso peptide biosynthesis component in a cell-free biosynthesis reaction mixture; and (b) incubating the cell-free biosynthesis reaction mixture under a condition suitable for lasso formation to produce the engineered lasso peptide; wherein the lasso peptide biosynthesis component comprises a lasso peptidase capable of catalyzing removal of the leader sequence. Particularly, in these embodiments, the corresponding leader sequence and lasso peptide biosynthesis components can be those disclosed in PCT application publication No.:WO2019/191571. In some embodiments, at least two of the first, second, third and fourth nucleic acid sequences are in a same nucleic acid molecule. In some embodiments, the engineered lasso peptide comprises an amino acid sequence as described herein (e.g, Table 1) and is G1-D9 cyclized. In some embodiments, the cell-free biosynthesis reaction mixture comprises cell extract or supplemented cell extract.

[00238] In some embodiments, cell-free biosynthesis of lasso peptides is conducted with isolated peptide and enzyme components in standard buffered media, such as phosphate- buffered saline or tris-buffered saline, in each case containing salts, ATP, and co-factors required for lasso peptidase and lasso cyclase enzymatic activity. In some embodiments, cell-free biosynthesis of lasso peptides is conducted using genes that require transcription (TX) and translation (TL) to afford the lasso precursor peptide and/or lasso peptide biosynthetic enzymes in situ, and such in vitro biosynthesis processes are conducted in cell extracts derived from prokaryotic or eukaryotic cells (See: Gagoski, D., et al., BiotechnoL Bioeng. 2016; 113 : 292-300; Culler, S. et al., PCT Appl. No. WO2017/031399).

[00239] In some embodiments, CFB reactions are conducted with a minimal set of lasso peptide biosynthesis components combined with genes that encode additional peptides, proteins or enzymes, including genes that encode RiPP recognition elements (RREs) or oligonucleotides that encode RREs that are fused to the 5’ or 3’ end of a lasso precursor peptide gene, an engineered lasso core peptide gene, a lasso peptidase gene or a lasso cyclase gene. In other embodiments, CFB reactions are conducted with a minimal set of lasso peptide biosynthesis components, including lasso precursor peptides, lasso peptidases, or lasso cyclase that are fused to RREs at the N-terminus or C-terminus. In other embodiments, CFB reactions are conducted with a minimal set of lasso peptide biosynthesis components combined and contacted with additional isolated proteins or enzymes, including RiPP recognition elements (RREs).

[00240] In some embodiments, CFB reactions are conducted with a minimal set of lasso peptide biosynthesis components combined and contacted with genes that encode additional proteins or enzymes, including genes that encode lasso peptide modifying enzymes such as N-methyltransferases, O-methyltransferases, biotin ligases, glycosyltransferases, esterases, acylases, acyltransferases, aminotransferases, amidases, hydroxylases, dehydrogenases, halogenases, kinases, RiPP heterocyclases, RiPP cyclodehydratases, peptidylarginine deiminase, and prenyltransferases.

[00241] In some embodiments, CFB reactions are conducted with a minimal set of lasso peptide biosynthesis components combined and contacted with additional isolated proteins or enzymes, including lasso peptide modifying enzymes such as N-methyltransferases, O- methyltransferases, biotin ligases, glycosyltransferases, esterases, acylases, acyltransferases, aminotransferases, amidases, hydroxylases, dehydrogenases, halogenases, kinases, RiPP heterocyclases, RiPP cyclodehydratases, peptidylarginine deiminase, and prenyltransferases. [00242] CFB methods and systems provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are conducted in a CFB reaction mixture, comprising one or more cell extracts that are supplemented with all twenty proteinogenic naturally occurring amino acids and corresponding transfer ribonucleic acids (tRNAs). Cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components also can be supplemented with additional components, including but not limited to, glucose, xylose, fructose, sucrose, maltose, starch, adenosine triphosphate (ATP), and/or adenosine diphosphate (ADP), purine and guanidine nucleotides, adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, and uridine triphosphate, cyclic-adenosine monophosphate (cAMP) and/or 3 -phosphoglyceric acid (3-PGA), nicotinamide adenine dinucleotides NADH and/or NAD, or nicotinamide adenine dinucleotide phosphates, NADPH, and/or NADP, or combinations thereof, amino acid salts such as magnesium glutamate and/or potassium glutamate, buffering agents such as HEPES, TRIS, spermidine, or phosphate salts, inorganic salts, including but not limited to, potassium phosphate, sodium chloride, magnesium phosphate, and magnesium sulfate, folinic acid and co-enzyme A (CoA), crowding agents such as PEG 8000, Ficoll 70, or Ficoll 400, L(-)-5-formyl-5,6,7,8- tetrahydrofolic acid, RNA polymerase, biotin, 1,4-dithiothreitol (DTT), magnesium acetate, ammonium acetate , or combinations thereof. For a general description of cell-free extract production and preparation, see: Krinsky, N., et al., PLoS ONE, 2016, 11(10): e0165137. [00243] In alternative embodiments, the preparation CFB reaction mixtures and cell extracts employed for the CFB methods as provided herein, comprises characterization of the CFB reaction mixtures and cell extracts using proteomic approaches to assess and quantify the proteome available for the production of lasso peptides and related molecules thereof. In alternative embodiments, ¹³C metabolic flux analysis (MFA) and/or metabolomics studies are conducted on CFB reaction mixtures and cell extracts to create a flux map and characterize the resulting metabolome of the CFB reaction mixture and cell extract or extracts.

[00244] In other embodiments, the CFB method is performed using: one or a combination of two or more cell extracts from various “chassis” organisms, such as E. coll, optionally mixed with one or a combination of two or more cell extracts derived from other species, e.g., a native lasso peptide-producing organism or relative. This can give the advantage of a robust transcription/translation machinery, combined with any unknown components of the native species that might be needed for proper protein folding or activity, or to supply precursors for the lasso peptide pathway. In alternative embodiments, if these factors are known they can be expressed in the chassis organism prior to making the cell extract or these factors can be isolated and purified and added directly to the CFB reaction mixture or cell extract.

[00245] In alternative embodiments, CFB methods and systems provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, including the use of cell extracts for in vitro TX-TL systems express lasso peptide biosynthetic gene clusters without the regulatory constraints of the cell. In alternative embodiments, some or all of the lasso peptide pathway biosynthetic genes are refactored to remove native transcriptional and translational regulation. In alternative embodiments, some or all of the lasso peptide pathway biosynthetic genes are refactored and constructed into operons on plasmids.

[00246] In alternative embodiments, CFB methods, systems and processes, including in vitro TX-TL systems, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are cell-free platforms that can use whole cell, cytoplasmic or nuclear extract from a single organism such as E.coli or Saccharomyces cerevisiae (S. cerevisiae) or from an organism of the Actinomyces genus, e.g., a Streptomyces. In alternative embodiments, CFB methods, systems and processes, including in vitro TX-TL systems, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are cell-free platforms that can use mixtures of whole cell, cytoplasmic, and/or nuclear extracts from the same or different organisms. In alternative embodiments, strain engineering approaches as well as modification of the growth conditions are used (on the organism from which at least one extract is derived) towards the creation of cell extracts as provided herein, to generate mixed cell extracts with varying proteomic and metabolic capabilities in the final CFB reaction mixture. In alternative embodiments, both approaches are used to tailor or design a final CFB reaction mixture for the purpose of synthesizing and characterizing lasso peptides, or for the creation of lasso peptide analogs through combinatorial biosynthesis approaches.

[00247] In alternative embodiments, cell extracts used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, comprise whole cell, cytoplasmic or nuclear extracts from a bacterial cell or eukaryotic cell, including insect, plant, fungal, yeast, or mammalian cells. In alternative embodiments, cell extracts used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, comprise whole cell, cytoplasmic or nuclear extracts from a bacterial cell or eukaryotic cell, including insect, plant, fungal, yeast, or mammalian cells, and are designed, produced and processed in a way to maximize efficacy and yield in the production of desired lasso peptides or related molecules thereof.

[00248] In an alternative embodiment, cell extracts used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, derive from at least two different bacterial cells, two different fungal cells; two different yeast cells, two different insect cells, two different plant cells or two different mammalian cells, or combinations of cell extracts from different species and genera thereof. In alternative embodiments, cell extracts used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, comprises an extract derived from: an Escherichia or a Escherichia coli (E. coli) a Streptomyces or an Aclinobacleria: an Ascomycota, Basidiomycota, or a Saccharomycelales a Penicillium or a Trichocomaceae: a Spodoptera, a Spodoptera frugiperda, a Trichoplusia or a Trichoplusia ni; a Poaceae, a Triticum, or a wheat germ; a rabbit reticulocyte or a HeLa cell.

[00249] In alternative embodiments, cell extracts used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, comprises a cell extract from or comprises an extract derived from: any prokaryotic and eukaryotic organism including, but not limited to, bacteria, including Archaea, eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human cells. In alternative embodiments, at least one of the cell extracts used in the CFB methods provided herein comprises an extract from or comprises an extract derived from: Escherichia coli, Saccharomyces cerevisiae, Saccharomyces kluyveri, Candida boidinii, Clostridium kluyveri, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium difficile, Clostridium botulinum, Clostridium tyrobutyricum, Clostridium tetanomorphum, Clostridium tetani, Clostridium propionicum, Clostridium aminobutyricum, Clostridium subterminale, Clostridium sticklandii, Ralstonia eutropha, Mycobacterium bovis, Mycobacterium tuberculosis, Porphyromonas gingivalis, Arabidopsis thaliana, Thermus thermophilus, Pseudomonas species, including Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, Homo sapiens, Oryctolagus cuniculus, Rhodobacter spaeroides, Thermo-anaerobacter brockii, Metallosphaera sedula, Leuconostoc mesenteroides, Chloroflexus aurantiacus, Roseiflexus castenholzii, Erythrobacter, Simmondsia chinensis, Acinetobacter species, including Acinetobacter calcoaceticus and Acinetobacter baylyi, Porphyromonas gingivalis, Sulfolobus tokodaii, Sulfolobus solfataricus, Sulfolobus acidocaldarius, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus brevis, Bacillus pumilus, Rattus norvegicus, Klebsiella pneumonia, Klebsiella oxytoca, Euglena gracilis, Treponema denticola, Moorella thermoacetica, Thermotoga maritima, Halobacterium salinarum, Geobacillus stearothermophilus, Aeropyrum pernix, Sus scrofa, Caenorhabditis elegans, Corynebacterium glutamicum, Acidaminococcus fermentans, Lactococcus lactis, Lactobacillus plantarum, Streptococcus thermophilus, Enterobacter aerogenes, Candida, Aspergillus terreus, Pedicoccus pentosaceus, Zymomonas mobilus, Acetobacter pasteurians, Kluyveromyces lactis, Eubacterium barkeri, Bacteroides capillosus, Anaerotruncus colihominis, Natranaerobius thermophilusm, Campylobacter jejuni, Haemophilus influenzae, Serratia marcescens, Citrobacter amalonaticus, Myxococcus xanthus, Fusobacterium nuleatum, Penicillium chrysogenum, marine gamma proteobacterium, butyrate -producing bacterium, Nocardia iowensis, Nocardia farcinica, Streptomyces griseus, Schizosaccharomyces pombe, Geobacillus thermoglucosidasius, Salmonella typhimurium, Vibrio cholera, Heliobacter pylori, Nicotiana tabacum, Oryza sativa, Haloferax mediterranei, Agrobacterium tumefaciens, Achromobacter denitrificans, Fusobacterium nucleatum, Streptomyces clavuligenus, Acinetobacter baumanii, Mus musculus, Lachancea kluyveri, Trichomonas vaginalis, Trypanosoma brucei, Pseudomonas stutzeri, Bradyrhizobium japonicum, Mesorhizobium loti, Bos taurus, Nicotiana glutinosa, Vibrio vulnificus, Vibrio natriegens, Selenomonas ruminantium, Vibrio parahaemolyticus, Archaeoglobus fulgidus, Haloarcula marismortui, Pyrobaculum aerophilum, Mycobacterium smegmatis MC2 155, Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium marinum M, Tsukamurella paurometabola DSM 20162, Cyanobium PCC7001, Dictyostelium discoideum AX4.

[00250] In alternative embodiments, at least one cell, cytoplasmic or nuclear extract used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, comprises a cell extract from or comprises an extract derived from: Acinetobacter baumannii Naval-82, Acinetobacter sp. ADP1, Acinetobacter sp. strain M-1, Actinobacillus succinogenes 130Z, Allochromatium vinosum DSM 180, Amycolatopsis methanolica, Arabidopsis thaliana, Atopobium parvulum DSM 20469, Azotobacter vinelandii DJ, Bacillus alcalophilus ATCC 27647, Bacillus azotoformans LMG 9581, Bacillus coagulans 36D1, Bacillus megaterium, Bacillus methanolicus MGA3, Bacillus methanolicus PB1, Bacillus methanolicus PB-1, Bacillus selenitireducens MLS 10 , Bacillus smithii, Bacillus subtilis , Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia multivorans, Burkholderia pyrrocinia, Burkholderia stabilis, Burkholderia thailandensis E264, Burkholderiales bacterium Joshi 001, Butyrate-producing bacterium L2-50, Campylobacter jejuni, Candida albicans, Candida boidinii, Candida methylica, Carboxydothermus hydrogenof ormans, Carboxydothermus hydrogenoformans Z-2901, Caulobacter sp. AP07, Chloroflexus aggregans DSM 9485, Chloroflexus aurantiacus J- 10-fl, Citrobacter freundii, Citrobacter koseri ATCC BAA-895, Citrobacter youngae , Clostridium, Clostridium acetobutylicum, Clostridium acetobutylicum ATCC 824, Clostridium acidurici, Clostridium aminobutyricum, Clostridium asparagiforme DSM 15981, Clostridium beijerinckii , Clostridium beijerinckii NCIMB 8052, Clostridium bolteae ATCC BAA-613, Clostridium carboxidivorans P7, Clostridium cellulovorans 743B, Clostridium difficile, Clostridium hiranonis DSM 13275, Clostridium hylemonae DSM 15053, Clostridium kluyveri, Clostridium kluyveri DSM 555, Clostridium ljungdahli, Clostridium ljungdahlii DSM 13528, Clostridium methylpentosum DSM 5476 , Clostridium pasteurianum, Clostridium pasteurianum DSM 525, Clostridium perfringens, Clostridium perfringens ATCC 13124, Clostridium perfringens str. 13, Clostridium phytofermentans ISDg, Clostridium saccharobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium saccharoperbutylacetonicum N1-4, Clostridium tetani, Corynebacterium glutamicum ATCC 14067, Corynebacterium glutamicum R, Corynebacterium sp. U-96, Corynebacterium variabile, Cupriavidus necator N-1, Cyanobium PCC7001, Desulfatibacillum alkenivorans AK-01, Desulfitobacterium hafniense, Desulfitobacterium metallireducens DSM 15288, Desulfotomaculum reducens M1-1, Desulfovibrio africanus str. Walvis Bay, Desulfovibrio fructosovorans JJ, Desulfovibrio vulgaris str. Hildenborough, Desulfovibrio vulgaris str. Miyazaki F', Dictyostelium discoideum AX4, Escherichia coli, Escherichia coli K-12 , Escherichia coli K-12 MG 1655, Eubacterium hallii DSM 3353 , Flavobacterium frigoris, Fusobacterium nucleatum subsp. polymorphum ATCC 10953 , Geobacillus sp. Y4.1MC1, Geobacillus themodenitrificans NG80-2, Geobacter bemidjiensis Bern, Geobacter sulfurreducens, Geobacter sulfurreducens PCA, Geobacillus stearothermophilus DSM 2334, Haemophilus influenzae, Helicobacter pylori, Homo sapiens, Hydrogenobacter thermophilus, Hydrogenobacter thermophilus TK-6, Hyphomicrobium denitrificans ATCC 51888, Hyphomicrobium zavarzinii, Klebsiella pneumoniae, Klebsiella pneumoniae subsp. pneumoniae MGH 78578, Lactobacillus brevis ATCC 367, Leuconostoc mesenteroides, Lysinibacillus fusiformis, Lysinibacillus sphaericus, Mesorhizobium loti MAFF 303099, Metallosphaera sedula, Methanosarcina acetivorans, Methanosarcina acetivorans C2A, Methanosarcina barkeri, Methanosarcina mazei Tuc01, Methylobacter marinus, Methylobacterium extorquens, Methylobacterium extorquens AMI, Methylococcus capsulatas, Methylomonas aminofaciens, Moorella thermoacetica, Mycobacter sp. strain JC1 DSM 3803, Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium bovis BCG, Mycobacterium gastri , Mycobacterium marinum M, Mycobacterium smegmatis, Mycobacterium smegmatis MC2 155, Mycobacterium tuberculosis, Nitrosopumilus salaria BD31 , Nitrososphaera gar gensis Ga9.2, Nocardia farcinica IFM 10152, Nocardia iowensis (sp. NRRL 5646), Nostoc sp. PCC 7120, Ogataea angusta, Ogataea parapolymorpha DL-1 (Hansenula polymorpha DL-1), Paenibacillus peoriae KCTC 3763, Paracoccus denitrificans, Penicillium chrysogenum, Photobacterium profitndum 3TCK, Phytofermentans ISDg, Pichia pastoris, Pier ophilus torridus DSM9790, Porphyromonas gingivalis, Porphyromonas gingivalis W83, Pseudomonas aeruginosa PA01, Pseudomonas denitrificans, Pseudomonas knackmussii, Pseudomonas putida, Pseudomonas sp, Pseudomonas syringae pv. syringae B728a, Pyrobaculum islandicum DSM 4184, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii OT3, Ralstonia eutropha, Ralstonia eutropha Hl 6, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodobacter sphaeroides ATCC 17025, Rhodopseudomonas palustris, Rhodopseudomonas palustris CGA009, Rhodopseudomonas palustris DX-1, Rhodospirillum rubrum, Rhodospirillum rubrum ATCC 11170, Ruminococcus obeum ATCC 29174, Saccharomyces cerevisiae, Saccharomyces cerevisiae S288c, Salmonella enterica, Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, Salmonella enterica typhimurium , Salmonella typhimurium, Schizosaccharomyces pombe, Sebaldella termitidis ATCC 33386 , Shewanella oneidensis MR-1, Sinorhizobium meliloti 1021, Streptomyces coelicolor, Streptomyces griseus subsp. griseus NBRC 13350, Sulfolobus acidocalarius, Sulfolobus solfataricus P-2, Synechocystis str. PCC 6803, Syntrophobacter fumaroxidans, Thauera aromatica, Thermoanaerobacter sp. X514, Thermococcus kodakaraensis, Thermococcus litoralis, Thermoplasma acidophilum, Thermoproteus neutrophilus, Thermotoga maritima, Thiocapsa roseopersicina, Tolumonas auensis DSM 9187, Trichomonas vaginalis G3, Trypanosoma brucei, Tsukamurella paurometabola DSM 20162, Vibrio cholera, Vibrio harveyi ATCC BAA-1116, Vibrio natriegens, Xanthobacter autotrophicus Py2, Yersinia intermedia, or Zea mays.

[00251] In alternative embodiments, cell extracts used in the CFB methods and processes, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, e.g., including at least one of the cell, cytoplasmic or nuclear extracts, have added to them, or further comprise, supplemental ingredients, compositions or compounds, reagents, ions, trace metals, salts, or elements, buffers and/or solutions. In alternative embodiments, the CFB method and system of the present disclosure, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, use or fabricate environmental conditions to optimize the rate of formation or yield of a lasso peptide or related molecules thereof.

[00252] In alternative embodiments, CFB reaction mixtures and cell extracts used in the CFB methods and systems, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with a carbon source and other essential nutrients. The CFB production system, including cell extracts used in the CFB methods and processes, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, can include, for example, any carbohydrate source. Such sources of sugars or carbohydrate substrates include glucose, xylose, maltose, arabinose, galactose, mannose, maltodextrin, fructose, sucrose and starch.

[00253] In alternative embodiments, CFB methods and systems provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are conducted in a CFB reaction mixture, comprising cell extracts that are supplemented with all twenty proteinogenic naturally occurring amino acids and corresponding transfer ribonucleic acids (tRNAs). In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with adenosine triphosphate (ATP), and/or adenosine diphosphate (ADP). In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with glucose, xylose, maltose, arabinose, galactose, mannose, maltodextrin, fructose, sucrose and/or starch. In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with purine and guanidine nucleotides, adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, and uridine triphosphate. In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with cyclic-adenosine monophosphate (cAMP) and/or 3 -phosphoglyceric acid (3-PGA). In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with nicotinamide adenine dinucleotides NADH and/or NAD, or nicotinamide adenine dinucleotide phosphates, NADPH, and/or NADP, or combinations thereof. In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with amino acid salts such as magnesium glutamate and/or potassium glutamate. In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with buffering agents such as HEPES, TRIS, spermidine, or phosphate salts. In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with salts, including but not limited to, potassium phosphate, sodium chloride, magnesium phosphate, and magnesium sulfate. In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with folinic acid and co-enzyme A (CoA). In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with crowding agents such as PEG 8000, Ficoll 70, or Ficoll 400, or combinations thereof. For a general description of cell-free extract production and preparation, see: Krinsky, N., et al., PLoS ONE, 2016, 11(10): e0165137. 7.4.3.3 Cell-based Biosynthesis of Engineered Lasso Peptides

[00254] In a related aspect, provided herein are non-naturally occurring microbial organisms having a recombinant nucleic acid or vector (e.g., expression vector) encoding an engineered lasso peptide described herein, as well as methods for producing engineered lasso peptides described herein using such non-naturally occurring microbial organisms. Certain cell-based production methods involve cultivating or fermenting such microbial organism that has been engineered to produce an engineered lasso peptide. Additionally, cell-based production methods can include cloning the genes encoding a lasso peptide biosynthesis component into an appropriate vector (e.g., expression vector), introducing that vector into a microorganism, and propagating or cultivating that organism with the necessary nutrients and under conditions for heterologous production of an engineered lasso peptide described herein (Zhang, Y., et al., Heterologous production of microbial ribosomally synthesized and post- translationally modified peptides, Front. Microbiol., 2018, doi: 10.3389/fmicb.2018.01801). [00255] Depending on the lasso peptide biosynthetic pathway constituents of a selected host microbial organism, in some embodiments, a non-naturally occurring microbial organism provided herein will include at least one exogenously expressed lasso peptide pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more lasso peptide biosynthetic pathways. For example, lasso peptide biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes or proteins of a lasso peptide pathway, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of a lasso peptide can be included, such as a lasso precursor peptide having an engineered lasso peptide amino sequence described herein, a lasso peptide peptidase, a lasso peptide cyclase, and/or a lasso peptide RiPP recognition element (RRE).

[00256] Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the lasso peptide pathway deficiencies of the selected host microbial organism. Therefore, a non-naturally occurring microbial organism of the disclosure can have one, two, three, four, five, six, seven, eight, nine or ten, up to all nucleic acids encoding the enzymes or proteins constituting a lasso peptide biosynthetic pathway disclosed herein. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize lasso peptide biosynthesis or that confer other useful functions onto the host microbial organism. One such other functionality can include, for example, augmentation of the synthesis of one or more of the lasso peptide pathway precursors, such as amino acids.

[00257] Generally, a host microbial organism is selected such that it produces the lasso precursor peptide, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host microbial organism. For example, amino acids are produced naturally in a host organism such as E. coli. A host organism can be engineered to increase production of a lasso precursor, as disclosed herein. In addition, a microbial organism that has been engineered to produce a desirable lasso precursor peptide can be used as a host organism and further engineered to express enzymes or proteins that processes the lasso precursor peptide into matured lasso peptides.

[00258] In some embodiments, a non-naturally occurring microbial organism described herein is generated from a host that contains the enzymatic capability to synthesize an engineered lasso peptide. In this specific embodiment it can be useful to increase the synthesis or accumulation of a lasso peptide pathway product to, for example, drive lasso peptide pathway reactions toward lasso peptide production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described lasso peptide pathway enzymes or proteins.

Overexpression of the enzyme or enzymes and/or protein or proteins of the lasso peptide pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes. Therefore, naturally occurring organisms can be readily generated to be non-naturally occurring microbial organisms described herein, for example, producing lasso peptide, through overexpression of one, two, three, four, five, six, seven, eight, nine, or ten, that is, up to all nucleic acids encoding a lasso peptide biosynthetic pathway enzymes or proteins. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the lasso peptide biosynthetic pathway.

[00259] In particularly useful embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene’s promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.

[00260] It is understood that, in methods described herein, any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non- naturally occurring microbial organism described herein. The nucleic acids can be introduced so as to confer, for example, a lasso peptide biosynthetic pathway onto the microbial organism. Alternatively, encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer lasso peptide biosynthetic capability. For example, a non- naturally occurring microbial organism having a lasso peptide biosynthetic pathway can comprise at least one exogenous nucleic acid encoding desired enzymes or proteins, such as the lasso precursor peptide, or alternatively a combination of a lasso peptide peptidase and a lasso peptide cyclase. Thus, it is understood that any combination of one or more genes encoding one or more peptides, enzymes, or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism described herein. Similarly, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism described herein, for example, lasso peptide peptidase and a lasso peptide cyclase, and so forth, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product.

[00261] In addition to the biosynthesis of engineered lasso peptides as described herein, the non-naturally occurring microbial organisms and methods described herein also can be utilized in various combinations with each other and with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes. For example, one alternative to produce an engineered lasso peptide other than use of the engineered lasso peptide producers is through addition of another microbial organism capable of converting a lasso peptide pathway intermediate to an engineered lasso peptide. One such procedure includes, for example, the fermentation of a microbial organism that produces a lasso precursor peptide. The lasso precursor peptide can then be used as a substrate for a second microbial organism that converts the lasso precursor peptide to an engineered lasso peptide. The lasso precursor peptide can be added directly to another culture of the second organism or the original culture of the lasso precursor peptide producers can be depleted of these microbial organisms by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps. Alternatively, an engineered lasso peptide also can be biosynthetically produced from microbial organisms through co-culture or co-fermentation using two organisms in the same vessel, where the first microbial organism produces a lasso precursor peptide and the second microbial organism converts the intermediate to an engineered lasso peptide. Alternatively, an engineered lasso peptide also can be biosynthetically produced by first chemically synthesizing the lasso precursor peptide, followed by addition of the chemically synthesized lasso precursor peptide to a fermentation broth using one or more organisms in the same vessel, where lasso precursor peptide is converted to an engineered lasso peptide. Alternatively, an engineered lasso peptide also can be biosynthetically produced from microbial organisms through cell-free biosynthesis of the lasso precursor peptide, followed by addition of the lasso precursor peptide fermentation broth using one or more organisms in the same vessel, where lasso precursor peptide is converted to an engineered lasso peptide. Alternatively, an engineered lasso peptide also can be biosynthetically produced by first chemically synthesizing the lasso precursor peptide, followed by addition of the chemically synthesized lasso precursor peptide to a broth containing the isolated biosynthetic enzymes, including but not limited to one or more of a lasso peptide peptidase, a lasso peptide cyclase, and lasso peptide RRE, wherein the lasso precursor peptide is converted to an engineered lasso peptide. Alternatively, an engineered lasso peptide also can be biosynthetically produced by first producing the lasso precursor peptide by cell-free biosynthesis methods, followed by addition of the lasso precursor peptide to a broth containing the isolated biosynthetic enzymes, including but not limited to one or more of a lasso peptide peptidase, a lasso peptide cyclase, and lasso peptide RRE, wherein the lasso precursor peptide is converted to an engineered lasso peptide. [00262] Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non-naturally occurring microbial organisms and methods described herein together with other microbial organisms, with the co-culture of other non-naturally occurring microbial organisms having sub-pathways and with combinations of other chemical and/or biochemical procedures well known in the art to produce an engineered lasso peptide.

[00263] In some embodiments, the microbial organisms comprises one or more fusion protein, or a polynucleotide encoding the fusion protein such that the microbial organism is capable of producing the fusion protein through in vivo transcription and translation (TX-TL) of the polynucleotide encoding the fusion protein.

[00264] In some embodiments, the fusion protein comprised a lasso precursor peptide or an engineered lasso core peptide fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide is fused at the C- terminus of the lasso precursor peptide or lasso core peptide. In some embodiments, a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the lasso precursor peptide or the lasso core peptide, wherein the 3’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide. In some embodiments, the fusion protein comprises an amino acid linker between the lasso precursor peptide or lasso core peptide and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not comprise an amino acid linker between the lasso precursor peptide or lasso core peptide and the one or more additional peptide or polypeptide.

[00265] In some embodiments, the fusion protein comprised a lasso precursor peptide or an engineered lasso core peptide fused to one or more lasso peptide biosynthesis components. In some embodiments, the one or more lasso peptide biosynthesis components are selected from (i) a lasso peptidase; (ii) a lasso cyclase; (iii) a RRE; or (iv) any combinations of (i) to (iii). In some embodiments, the one or more lasso peptide biosynthesis components are encoded by the same lasso peptide biosynthetic gene cluster. In other embodiments, the one or more lasso peptide biosynthesis components are encoded by different lasso peptide biosynthetic gene cluster.

[00266] In some embodiments, the fusion protein comprises an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not comprise an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide. [00267] In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso cyclase. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a RRE. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase and a lasso cyclase. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase and a RRE. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso cyclase and a RRE. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase, a lasso cyclase and RRE. In specific embodiments, the fusion protein comprises an engineered lasso core peptide fused to a lasso peptidase. In specific embodiments, the fusion protein comprises an engineered lasso core peptide fused to a lasso cyclase. In specific embodiments, the fusion protein comprises an engineered lasso core peptide fused to a RRE. In specific embodiments, the fusion protein comprises an engineered lasso core peptide fused to a lasso peptidase and a lasso cyclase. In specific embodiments, the fusion protein comprises an engineered lasso core peptide fused to a lasso peptidase and a RRE. In specific embodiments, the fusion protein comprises an engineered lasso core peptide fused to a lasso cyclase and a RRE. In specific embodiments, the fusion protein comprises an engineered lasso core peptide fused to a lasso peptidase, a lasso cyclase and RRE.

[00268] In some embodiments, the fusion protein comprised a lasso precursor peptide or an engineered lasso core peptide fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide comprises a peptide or polypeptide that facilitates production of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom through cell-free biosynthesis. Examples of peptide or polypeptide that can be fused with a lasso precursor peptide or an engineered lasso core peptide according to the present disclosure include but are not limited to (i) a peptide or polypeptide that increases the level of transcription of the lasso precursor peptide or lasso core peptide in the microbial organism; (ii) a peptide or polypeptide that increases the level of translation of the lasso precursor peptide or lasso core peptide in the microbial organism; (iii) a peptide or polypeptide that facilitates the processing of the lasso precursor peptide or lasso core peptide into the engineered lasso peptide; (iv) a peptide or polypeptide that improves stability of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; (v) a peptide or polypeptide that improves solubility of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; (vi) a peptide or polypeptide that enables or facilitates the detection of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; (vii) a peptide or polypeptide that enables or facilitates purification of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; (viii) a peptide or polypeptide that enables or facilitates immobilization of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; or (ix) any combination of (i) to (viii).

[00269] In some embodiments, the fusion protein comprised a lasso precursor peptide or an engineered lasso core peptide fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide comprises a biologically active peptide or polypeptide. Examples of biologically active peptide or polypeptide that can be fused with a lasso precursor peptide or lasso core peptide according to the present disclosure include but are not limited to (i) a peptide or polypeptide capable of binding to a target molecule (e.g., an antibody, an antigen, or a receptor); (ii) a peptide or polypeptide that enhance cell permeability of the fusion protein; (iii) a peptide or polypeptide capable of conjugating the fusion protein to at least one additional copy of the fusion protein; (iv) a peptide or polypeptide capable of linking the fusion protein to one or more peptidic or non-peptidic molecule; (v) a peptide or polypeptide capable of modulating activity of the lasso precursor peptide or lasso core peptide; (vi) a peptide or polypeptide capable of modulating activity of the engineered lasso peptide derived from the lasso precursor peptide or the lasso core peptide; or (vii) any combinations of (i) to (vi).

[00270] In some embodiments, the fusion protein comprised a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide is fused to the N-terminus of the lasso peptidase or the lasso cyclase. In some embodiments, the one or more additional peptide or polypeptide is fused at the C-terminus of the lasso peptidase or the lasso cyclase. In some embodiments, a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the lasso peptidase or the lasso cyclase, wherein the 5’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide. In some embodiments, a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the lasso peptidase or the lasso cyclase, wherein the 3’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide. In some embodiments, the fusion protein comprises an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not comprise an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide.

[00271] In some embodiments, the fusion protein comprised a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide. In some embodiments, the more additional peptide or polypeptide comprises a peptide or polypeptide encoded by a lasso peptide biosynthetic gene cluster. Examples of peptide or polypeptide that can be fused with a lasso precursor peptide or an engineered lasso core peptide according to the present disclosure include but are not limited to (i) a lasso precursor peptide; (ii) an engineered lasso core peptide; (iii) a lasso peptidase; (iv) a lasso cyclase, (v) a RRE; or (vi) any combinations of (i) to (vi). In specific embodiments, the fusion protein comprises at least one lasso cyclase and at least one lasso peptidase. In specific embodiments, the fusion protein comprises at least one lasso cyclase fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso peptidase fused to a RRE.

[00272] In some embodiments, the fusion protein comprised a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide comprises a peptide or polypeptide that facilitates production of the lasso peptidase or lasso cyclase through cell-free biosynthesis. Examples of peptide or polypeptide that can be fused with the lasso peptidase or lasso cyclase according to the present disclosure include but are not limited to (i) a peptide or polypeptide that increases the level of transcription of the lasso peptidase or lasso cyclase in the microbial organism; (ii) a peptide or polypeptide that increases the level of translation of the lasso peptidase or lasso cyclase in the microbial organism; (iii) a peptide or polypeptide that improves stability of the lasso peptidase or lasso cyclase; (vi) a peptide or polypeptide that improves solubility of the lasso peptidase or lasso cyclase; (v) a peptide or polypeptide that enables or facilitates the detection of the lasso peptidase or lasso cyclase; (vi) a peptide or polypeptide that enables or facilitates purification of the lasso peptidase or lasso cyclase; (vii) a peptide or polypeptide that enables or facilitates immobilization of the lasso peptidase or lasso cyclase; or (viii) any combination of (i) to (vii). [00273] In some embodiments, the fusion protein comprised a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide comprises a biologically active peptide or polypeptide. Examples of biologically active peptide or polypeptide that can be fused with a lasso peptidase or a lasso cyclase according to the present disclosure include but are not limited to (i) a peptide or polypeptide capable of modulating the reaction catalyzing activity of the lasso peptidase or lasso cyclase; (ii) a peptide or polypeptide capable of modulating target specificity of the lasso peptidase or lasso cyclase; (iii) an enzyme having the same or different enzymatic activity as the lasso peptidase or lasso cyclase; or any combination of (i) to (iii).

[00274] In some embodiments, the fusion protein comprised a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide is fused to the N-terminus of the RRE. In some embodiments, the one or more additional peptide or polypeptide is fused at the C-terminus of the RRE. In some embodiments, a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the RRE, wherein the 5’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide. In some embodiments, a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the RRE, wherein the 3’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide. In some embodiments, the fusion protein comprises an amino acid linker between the RRE and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not comprise an amino acid linker between RRE and the one or more additional peptide or polypeptide.

[00275] In some embodiments, the fusion protein comprised a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide. In some embodiments, the more additional peptide or polypeptide comprises a peptide or polypeptide encoded by a lasso peptide biosynthetic gene cluster. Examples of peptide or polypeptide that can be fused with a lasso precursor peptide or an engineered lasso core peptide according to the present disclosure include but are not limited to (i) a lasso precursor peptide; (ii) an engineered lasso core peptide; (iii) a lasso peptidase; (iv) a lasso cyclase, (v) a RRE; or (vi) any combinations of (i) to (vi). In specific embodiments, the fusion protein comprises at least one lasso precursor peptide fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso core peptide fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso cyclase fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso peptidase fused to a RRE.

[00276] In some embodiments, the fusion protein comprised a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide comprises a peptide or polypeptide that facilitates production of the RRE through cell-free biosynthesis. Examples of peptide or polypeptide that can be fused with the RRE according to the present disclosure include but are not limited to (i) a peptide or polypeptide that increases the level of transcription of the RRE in the microbial organism; (ii) a peptide or polypeptide that increases the level of translation of the RRE in the microbial organism; (iii) a peptide or polypeptide that improves stability of the RRE; (vi) a peptide or polypeptide that improves solubility of the RRE; (v) a peptide or polypeptide that enables or facilitates the detection of the RRE; (vi) a peptide or polypeptide that enables or facilitates purification of the RRE; (vii) a peptide or polypeptide that enables or facilitates immobilization of the RRE; or (viii) any combination of (i) to (vii).

[00277] In some embodiments, the fusion protein comprised a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide comprises a biologically active peptide or polypeptide. Examples of biologically active peptide or polypeptide that can be fused with a RRE according to the present disclosure include but are not limited to (i) a peptide or polypeptide capable of modulating the reaction catalyzing activity of the lasso peptidase or lasso cyclase; (ii) a peptide or polypeptide capable of modulating target specificity of the lasso peptidase or lasso cyclase; (iii) an enzyme having the same or different enzymatic activity as the lasso peptidase or lasso cyclase; or any combination of (i) to (iii).

[00278] In particular embodiments, the lasso precursor peptide genes are fused at the 5’- terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, such as sequences encoding maltose-binding protein (MBP) or small ubiquitin-like modifier protein (SUMO), which enhance the stability, solubility, and production of the desired products (Marblestone, J.G., el ct!.. Protein Sci, 2006, 15, 182-189). In particular embodiments, the lasso precursor peptides are fused at the C-terminus of the leader sequences to form conjugates with peptides or proteins, such as maltose-binding protein or small ubiquitin-like modifier protein, which enhance the stability, solubility, and production of the fused MBP-lasso or SUMO-lasso precursor peptide. [00279] In particular embodiments, the lasso precursor peptide genes or lasso core peptide genes are fused at the 3 ’-terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, such as sequences encoding maltose-binding protein (MBP) or small ubiquitin-like modifier protein (SUMO), which enhance the stability, solubility, and production of the desired products. In particular embodiments, the lasso precursor peptides, lasso core peptides, or lasso peptides are fused at the N-terminus to form conjugates with peptides or proteins, such as maltose-binding protein or small ubiquitin-like modifier protein, which enhance the stability, solubility, and production of the fused MBP- lasso or SUMO-lasso precursor peptide.

[00280] In particular embodiments, the lasso precursor peptide genes or lasso core peptide genes are fused at the 5'-terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, with or without a linker, such as sequences encoding peptide tags for affinity purification or immobilization, including his-tags, strep- tags, or FLAG-tags. In some embodiments, the lasso precursor peptides, lasso core peptides, or lasso peptides are fused at the C-terminus of the core peptides to form conjugates with other peptides or proteins, with or without a linker, such as peptide tags for affinity purification or immobilization, including his-tags, strep-tags, or FLAG-tags.

[00281] In particular embodiments, lasso precursor peptides, lasso core peptides, or lasso peptides are fused to molecules that can enhance cell permeability or penetration into cells, for example through the use of arginine-rich cell-penetrating peptides such as TAT peptide, penetratin, and flock house virus (FHV) coat peptide (Brock, R., Bioconjug. Chem., 2014, 25, 863-868). In particular embodiments, a lasso precursor peptide gene or core peptide gene is fused at the 3 ’-terminus to oligonucleotide sequences that encode arginine-rich cell- penetrating peptides or proteins, including oligonucleotide sequences that encode penetratin, and flock house virus (FHV) coat peptide or similar peptides that contain guanidinium groups or a combination of lysine and guanidinium groups (Wender, P.A., el al., Adv. Drug Deliv. Rev., 2008, 60, 452-472). In particular embodiments, a lasso precursor peptide, lasso core peptide, or lasso peptide is fused at the C-terminus to peptides that promote cell penetration such as arginine-rich cell-penetrating peptides or proteins, including amino acid sequences that encode TAT peptide, penetratin, and flock house virus (FHV) coat peptide or similar peptides that contain guanidinium groups or a combination of lysine and guanidinium groups. [00282] In particular embodiments, the lasso precursor peptide genes or lasso core peptide genes are fused at the 5'-terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, with or without a linker, such as sequences encoding peptide epitopes that are known to bind with high affinity to antibodies, cell surface proteins, or cell surface receptors, including cytokine binding epitopes, integrin ligand binding epitopes, and the like. In particular embodiments, the lasso precursor peptides, lasso core peptides, or lasso peptides are fused at the C-terminus to peptides or proteins, with or without a linker, such as peptide epitopes that are known to bind with high affinity to antibodies, cell surface proteins, or cell surface receptors, including cytokine binding epitopes, integrin ligand binding epitopes, and the like.

[00283] In specific embodiments, the method comprises (a) introducing into the microbial organism a first nucleic acid sequence encoding an engineered lasso peptide described herein (e.g., Table 1) and a second nucleic acid sequence encoding a lasso peptide biosynthesis component; and (b) culturing the microbial organism under a condition suitable for lasso formation to produce the engineered lasso peptide. In some embodiments, the method comprises (a) introducing into the microbial organism a first nucleic acid sequence encoding an engineered lasso peptide described herein (e.g., Table 1) and a second nucleic acid sequence encoding a lasso peptide biosynthesis component comprising a lasso cyclase; and (b) culturing the microbial organism under a condition suitable for lasso formation to produce the engineered lasso peptide. In some embodiments, the method comprises (a) introducing into the microbial organism a first nucleic acid sequence encoding a lasso precursor peptide having an amino acid sequence as described herein (e.g., Table 1) and a leader sequence comprising the amino acid sequence of MTEQSEQTPTEYIPPMLVEVGEFTEDTL (SEQ ID NO: 118) and a second nucleic acid sequence encoding a lasso peptide biosynthesis component comprising a lasso peptidase and a lasso cyclase; and (b) culturing the microbial organism under a condition suitable for lasso formation to produce the engineered lasso peptide. In some embodiments, the engineered lasso peptide comprises an amino acid sequence as described herein (e.g., Table 1) and is G1-D9 cyclized.

[00284] In specific embodiments, the lasso peptide biosynthesis component comprises a lasso cyclase. In those embodiments where the lasso peptide biosynthesis component comprises a lasso peptidase and a lasso cyclase, the method comprises introducing the second nucleic acid sequence encoding the lasso cyclase and a third nucleic acid sequence encoding the lasso peptidase. In those embodiments where the lasso peptide biosynthesis component comprises a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE), the method comprises introducing the second nucleic acid sequence encoding the lasso cyclase and a fourth nucleic acid sequence encoding the RRE. In those embodiments where the lasso peptide biosynthesis component comprises a lasso peptidase, a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE), and the method comprises introducing the second nucleic acid sequence encoding the lasso cyclase, a third nucleic acid sequence encoding the lasso peptidase, and a fourth nucleic acid sequence encoding the RRE. In some embodiments, at least two of the first, second, third and fourth nucleic acid sequences are in a same nucleic acid molecule. In some embodiments, the engineered lasso peptide comprises an amino acid sequence as described herein (e.g., Table 1) and is G1-D9 cyclized.

[00285] In some embodiments, to produce engineered lasso peptides having amino acid sequences as described herein (e.g., Table 1), the core peptide sequences can be fused to any known leader sequence, thereby producing a lasso precursor peptide, and the method then employs one or more lasso peptide biosynthesis component capable of recognizing such leader sequence and processing the lasso precursor peptide into matured engineered lasso peptides having an amino acid sequence as described herein (e.g., Table 1).

[00286] Accordingly, in some embodiments, provided herein is a method of producing an engineered lasso peptide having an amino acid sequence as described herein (e.g., Table 1), wherein the method comprises (a) introducing into a microbial organism a first nucleic acid sequence encoding a lasso precursor peptide comprising a leader sequence and an engineered lasso core peptide sequence as described herein (e.g., Table 1) and a second nucleic acid sequence encoding a lasso peptide biosynthesis component; and (b) culturing the microbial organism under a condition suitable for lasso formation to produce the engineered lasso peptide; wherein the lasso peptide biosynthesis component comprises a lasso peptidase capable of catalyzing removal of the leader sequence. Particularly, in these embodiments, the corresponding leader sequence and lasso peptide biosynthesis components can be those disclosed in PCT application publication number: WO2019/191571. In some embodiments, at least two of the first, second, third and fourth nucleic acid sequences are in a same nucleic acid molecule. In some embodiments, the engineered lasso peptide comprises an amino acid sequence as described herein (e.g., Table 1) and is G1-D9 cyclized.

[00287] Any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the engineered lasso peptide described herein. For example, the engineered lasso peptide producers can be cultured for the biosynthetic production of engineered lasso peptide. For example, host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable to fermentation processes. Exemplary bacteria include species selected from Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Streptomyces venezuelae, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Streptomyces albus, Clostridium acetobutylicum, Streptomyces lividans, Vibrio natriegens, Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts or fungi include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, and the like. E. coli is a particularly useful host organisms since it is a well characterized microbial organism suitable for genetic engineering. Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae. Other particularly useful microbial organisms for the cell-based biosynthesis of engineered lasso peptides include, for example, Vibrio natriegens, Burholderia spp., Corynebacterium glutamicum, or Sphingomaons subterranean.

[00288] Sources of encoding nucleic acids for a lasso peptide pathway enzyme or protein can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction. Such species include both prokaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, Exemplary species for such sources include, for example, Escherichia coli, Acidaminococcus fermentans, Acinetobacter baylyi, Acinetobacter calcoaceticus, Acinetobacter sp. ADP1, Acinetobacter sp. Strain M-l, Aquifex aeolicus, Arabidopsis thaliana, Arabidopsis thaliana col, Arabidopsis thaliana col, Archaeoglobus fulgidus DSM 4304, Azoarcus sp. CIB, Bacillus cereus, Bacillus subtilis, Bos Taurus, Brucella melitensis, Burkholderia ambifaria AMMD, Burkholderia phymatum, Campylobacter jejuni, Chlor oflexus aurantiacus, Citrobacter youngae ATCC 29220, Clostridium acetobutylicum, Clostridium aminobutyricum, Clostridium beijerinckii, Clostridium beijerinckii NCIMB 8052, Clostridium beijerinckii NRRL B593, Clostridium botulinum C str. Eklund, Clostridium kluyveri, Clostridium kluyveri DSM 555, Clostridium novyi NT, Clostridium propionicum, Clostridium saccharoperbutylacetonicum, Corynebacterium glutamicum ATCC 13032, Cupriavidus taiwanensis, Cyanobium PCC7001, Dictyostelium discoideum AX4, Enterococcus faecalis, Erythrobacter sp. NAP1, Escherichia coli K12, Escherichia coli str. K-12 substr. MG1655, Eubacterium rectale ATCC 33656, Fusobacterium nucleatum, Fusobacterium nucleatum subsp. nucleatum ATCC 25586, Geobacillus thermoglucosidasius, Haematococcus pluvialis, Haemophilus influenzae, Haloarcula marismortui ATCC 43049, Helicobacter pylori, Homo sapiens, Klebsiella pneumoniae, Lactobacillus plantarum, Leuconostoc mesenteroides, marine gamma proteobacterium HTCC2080, Metallosphaera sedula, Methanocaldococcus jannaschii, Mus musculus, Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium bovis BCG, Mycobacterium marinum M, Mycobacterium smegmatis MC2 155, Mycobacterium tuberculosis, Mycoplasma pneumoniae Ml 29, Nocardia farcinica IFM 10152, Nocardia iowensis (sp. NRRL 5646), Oryctolagus cuniculus, Paracoccus denitrificans, Penicillium chrysogenum, , Porphyromonas gingivalis, Porphyromonas gingivalis W83, Pseudomonas aeruginosa, Pseudomonas aeruginosa PAO1 , Pseudomonas fluorescens, Pseudomonas fluorescens Pf-5, Pseudomonas knackmussii (Bl 3), Pseudomonas putida, Pseudomonas putida E23, Pseudomonas putida KT2440, Pseudomonas sp, Pyrobaculum aerophilum str. IM2, Pyrococcus furiosus, Ralstonia eutropha, Ralstonia eutropha H16, Ralstonia eutropha Hl 6, Ralstonia metallidurans, Rattus norvegicus, Rhodobacter spaeroides, Rhodococcus rubber, Rhodopseudomonas palustris, Roseburia intestinalis Ll-82, Roseburia inulinivorans DSM 16841, Roseburia sp. A2-183, Roseiflexus castenholzii, NRRL 2338, Salmonella enterica subsp. arizonae serovar, Salmonella typhimurium, Schizosaccharomyces pombe, Simmondsia chinensis, Sinorhizobium meliloti, Staphylococcus aureus, Streptococcus pneumoniae, Streptomyces coelicolor, Streptomyces griseus subsp. griseus , BRC 13350, Streptomyces sp. ACT-1, Sulfolobus acidocaldarius, Sulfolobus shibatae, Sulfolobus solfataricus, Sulfolobus tokodaii, Synechocystis sp. strain PCC6803, Syntrophus , ciditrophicus, Thermoanaerobacter brockii HTD4, Thermoanaerobacter tengcongensis MB4, Thermosynechococcus elongates, Thermotoga maritime MSB8, Thermus thermophilus, Thermus, hermophilus HB8, Trichomonas vaginalis G3, Trichosporonoides megachiliensis, Trypanosoma brucei, Tsukamurella paurometabola DSM 20162, Vibrio natriegens, Yersinia intermedia ATCC 29909, Zoogloea ramigera, Zygosaccharomyces rouxii, Zymomonas mobilis, as well as other exemplary species disclosed herein are available as source organisms for corresponding genes. However, with the complete genome sequence available for now more than 100,000 bacterial species, the identification of genes encoding the requisite lasso peptide biosynthetic activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations allowing biosynthesis of an engineered lasso peptide described herein with reference to a particular organism such as E. coll can be readily applied to other microorganisms. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.

[00289] In some instances, such as when an alternative lasso peptide biosynthetic pathway exists in an unrelated species, engineered lasso peptide biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods of the disclosure can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize an engineered lasso peptide.

[00290] Methods for constructing and testing the expression levels of a non-naturally occurring lasso peptide-producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

[00291] Exogenous nucleic acid sequences involved in a pathway for production of an engineered lasso peptide can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the active proteins. [00292] An expression vector or vectors can be constructed to include one or more lasso peptide biosynthetic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms described herein include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

[00293] For the production of engineered lasso peptide, the recombinant strains are cultured in a medium with carbon source and other essential nutrients. It is sometimes desirable and can be highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well- known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in United State publication 2009/0047719, filed August 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein.

[00294] The growth medium can include, for example, any amino acid source, including essential amino acids that cannot be synthesized by the non-naturally occurring microbial organism or amino acids that stimulate cell growth, and/or a carbohydrate source that can supply a source of carbon to the non-naturally occurring microorganism. Such amino acid sources include, for example, beef or yeast extracts Such carbohydrate sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods described herein include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms described herein for the production of an engineered lasso peptide.

[00295] In various embodiments, the cell-free or cell-based biosynthesis system (e.g., a CFB reaction mixture of cell culture) can be maintained under aerobic conditions, where such conditions can be achieved, for example, by sparging with air or oxygen, shaking under an atmosphere of air or oxygen, stirring under an atmosphere of air or oxygen, or combinations thereof. Additionally, the cell-free or cell-based biosynthesis system (e.g., a CFB reaction mixture of cell culture) can be maintained under glucose conditions, where such conditions can be achieved, for example, by limiting the amount of amino acid and/or glucose that is added to the system, or combinations thereof.

[00296] In alternative embodiments, the cell-free or cell-based biosynthesis system (e.g., a CFB reaction mixture of cell culture) can be maintained under anaerobic or substantially anaerobic conditions, where such conditions can be achieved, for example, by first sparging the medium with nitrogen and then sealing the wells or reaction containers, or by shaking or stirring under a nitrogen atmosphere. Briefly, anaerobic conditions refer to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, biosynthesis processes conducted such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. Substantially anaerobic conditions also include performing the biosynthesis methods and processes inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the CFB reaction or cell culture with an N2/CO2 mixture or other suitable non-oxygen gas or gases.

[00297] If desired, the pH of the cell culture medium or CFB reaction mixture, including cell extracts, used in the biosynthesis methods and systems, provided herein for the synthesis of engineered lasso peptides and related molecules thereof can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a buffer, a base, such as NaOH or other bases, or an acid, as needed to maintain the production system at a desirable pH for high rates and yields in the production of engineered lasso peptides and related molecules thereof.

[00298] In alternative embodiments, the cell culture medium or CFB reaction mixture, including cell extracts, used in the CFB methods and systems, provided herein for the synthesis of engineered lasso peptides and related molecules thereof can be supplemented with one or more enzymes (or the nucleic acids that encode them) of central metabolism pathways, for example, one or more (or all of the) central metabolism enzymes from the tricarboxylic acid cycle (TCA, or Krebs cycle), the glycolysis pathway or the Citric Acid Cycle, or enzymes that promote the production of amino acids.

[00299] Metabolic modeling and simulation algorithms can be utilized to optimize conditions for the present biosynthesis process and to optimize engineered lasso peptide production rates and yields in the cell-free or cell-based system. Modeling can also be used to design gene knockouts that additionally optimize utilization of the lasso peptide pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on shifting the primary metabolism towards more efficient production of engineered lasso peptides and related molecules thereof.

[00300] One computational method for identifying and designing metabolic alterations favoring biosynthesis of a desired product is the OptKnock computational framework (Burgard et al., Biotechnol. Bioeng., 2003, 84, 647-657). OptKnock is a metabolic modeling and simulation program that suggests gene deletion or disruption strategies that result in genetically stable metabolic network which overproduces the target product. Specifically, the framework examines the complete metabolic and/or biochemical network in order to suggest genetic manipulations that lead to maximum production of an engineered lasso peptide or related molecules thereof. Such genetic manipulations can be performed on strains used to produce cell extracts for the CFB methods and processes provided herein. Also, this computational methodology can be used to either identify alternative pathways that lead to biosynthesis of a desired engineered lasso peptide or used in connection with non-naturally occurring systems for further optimization of biosynthesis of a desired engineered lasso peptide.

[00301] Briefly, OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism. The OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data. OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or deletions. OptKnock computational framework allows the construction of model formulations that allow an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems. The metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, U.S. publication 2002/0168654, filed January 10, 2002, in International Patent No. PCT/US02/00660, filed January 10, 2002, and U.S. publication 2009/0047719, filed August 10, 2007.

[00302] Another computational method for identifying and designing metabolic alterations favoring biosynthetic production of a product is a metabolic modeling and simulation system termed SimPheny®. This computational method and system is described in, for example, U.S. publication 2003/0233218, filed June 14, 2002, and in International Patent Application No. PCT/US03/18838, filed June 13, 2003. SimPheny® is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system. This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components.

[00303] These computational approaches are consistent with biological realities because biological systems are flexible and can reach the same result in different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, thereby enhancing the precision with which biosynthetic performance can be predicted.

[00304] Given the teachings and guidance provided herein, those skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement biosynthesis of engineered lasso peptides or related molecules thereof using whole cells or cell extracts and the biosynthesis methods and processes provided herein for the production of engineered lasso peptides and related molecules thereof. Such metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny® and OptKnock. Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods well known in the art.

[00305] Suitable purification and/or assays to test for the production of engineered lasso peptides can be performed using well known methods. Suitable replicates such as triplicate CFB reactions or cell cultures, can be conducted and analyzed to verify engineered lasso peptide production and concentrations. The final product of engineered lasso peptides can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectrometry), LC-MS (Liquid Chromatography-Mass Spectrometry), MALDI or other suitable analytical methods using routine procedures well known in the art. Byproducts and residual amino acids or glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and saturated fatty acids, and a UV detector for amino acids and other organic acids (Lin el al., Biotechnol. Bioeng., 2005, 90, 775-779), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities encoded by exogenous or endogenous DNA sequences can also be assayed using methods well known in the art.

[00306] Biosynthesized peptide or polypeptide can be isolated, separated purified from other components in the CFB reaction mixtures or cell culture medium using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures, including extraction of CFB reaction mixtures using organic solvents such as methanol, butanol, ethyl acetate, and the like, as well as methods that include continuous liquid-liquid extraction, solid-liquid extraction, solid phase extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, dialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, ultrafiltration, medium pressure liquid chromatograpy (MPLC), and high pressure liquid chromatography (HPLC). All of the above methods are well known in the art and can be implemented in either analytical or preparative modes.

7.4.4 Pharmaceutical Compositions

[00307] In a related aspect of the present disclosure, provided herein are pharmaceutical compositions containing an engineered lasso peptide described in Section 5.4.1, In some embodiments, the pharmaceutical composition contains an engineered lasso peptides that was biosynthesized using a method described in Section 5.4.3,

[00308] In some embodiments, the pharmaceutical composition contains an effective amount of at least one engineered lasso peptide and pharmaceutically acceptable carrier(s) or excipient(s). In some embodiments, the pharmaceutical composition further comprises an effective amount of at least one additional therapeutic agent that is not a lasso peptide. Such a therapeutic agent, in some embodiments, can be for managing, preventing or treating cancer, including, for example, a chemotherapy or immunotherapy.

[00309] In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, such as one or more of cyclophosphamide, thiotepa, mechlorethamine (chlormethine/mustine), uramustine, melphalan, chlorambucil, ifosfamide, chlomaphazine, cholophosphamide, estramustine, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, bendamustine, busulfan, improsulfan, piposulfan, carmustine, lomustine, chlorozotocin, fotemustine, nimustine, ranimustine, streptozucin, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, dacarbazine, mitozolomide, temozolomide, paclitaxel, docetaxel, vinblastine, vincristine, vinorelbine, cabazitaxel, dactinomycin (actinomycin D), calicheamicin, dynemicin, amsacrine, doxarubicin, daunorubicin, epirubicin, mitoxantrone, idarubicin, pirarubicin, benzodopa, carboquone, meturedopa, uredopa, altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, callystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancrati statin, sarcodictyin, spongistatin, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomysin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, detorubicin, 6-diazo- 5-oxo-L-norleucine, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, methotrexate, 5- fluorouracil (5-FU), denopterin, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, mitotane, trilostane, frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformithine, elliptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, PSK polysaccharide complex, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2, 2', 2”- trichlorotri ethylamine; T-2 toxin, verracurin A, roridin A and anguidine, urethan, vindesine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (“Ara-C”), etoposide (VP- 16), vinorelbine, novantrone, teniposide, edatrexate, aminopterin, xeloda, ibandronate, irinotecan (e.g., CPT-11), topoisomerase inhibitor RFS 2000, difluorometlhylornithine (DMFO), retinoic acid, capecitabine, plicomycin, gemcitabine, navelbine, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above. In specific embodiments, the chemotherapeutic agent comprises cyclophosphamide.

[00310] In some embodiments, the additional therapeutic agent can be a immunotherapeutic agent, such as one or more of immune checkpoint modulator that inhibits, decreases or interferes with the activity of a negative checkpoint regulator. In certain embodiments, the negative checkpoint regulator is selected from Cytotoxic T-lymphocyte antigen-4 (CTLA-4), CD40, CD47, CD80, CD86, Programmed cell death 1 (PD-1), Programmed cell death ligand 1 (PD-L1), Programmed cell death ligand 2 (PD-L2), Lymphocyte activation gene-3 (LAG-3; also known as CD223), Galectin-3, B and T lymphocyte attenuator (BTLA), T-cell membrane protein 3 (TIM3), Galectin-9 (GAL9), B7- Hl, B7-H3, B7-H4, T-Cell immunoreceptor with Ig and ITIM domains (TIGIT/Vstm3/WUCAM/VSIG9), V-domain Ig suppressor of T-Cell activation (VISTA), Glucocorticoid-induced tumor necrosis factor receptor-related (GITR) protein, Herpes Virus Entry Mediator (HVEM), 0X40, CD27, CD28, CD137. CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049, CGEN-15052, and CGEN-15092. In some embodiments, the immune checkpoint modulator is an anti -PD-1 antibody. In some embodiments, the immune checkpoint modulator is an anti-PD-Ll antibody.

[00311] In some embodiments, the additional therapeutic agent can be a cancer vaccine, such as sipuleucel-T vaccine, Bacillus Calmette-Guerin vaccine, LLO-E7 DNA vaccine, and T-VEC (Imlygic®).

[00312] The pharmaceutical compositions provided herein can be formulated for administration via a suitable route of administration, for example, for oral, nasal, subcutaneous, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intra-arterial, intravenous, intramuscular, or other parental routes of administration. Depending upon the selection of a particular route of administration, pharmaceutically-acceptable carriers well-known in the art can be used.

[00313] The pharmaceutical compositions provided herein can be formulated in a pharmaceutically acceptable formulation forms. Selection of a proper formulation of a pharmaceutical composition can depend the route of administration chosen. A summary of pharmaceutical compositions is found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins, 1999).

[00314] For example, suitable forms of formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast smelt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multi- particulate formulations, and mixed immediate and controlled release formulations. The pharmaceutical compositions will include at least one lasso peptide, as an active ingredient in free-acid or free-base form, or in a pharmaceutically acceptable salt form. In addition, the methods and pharmaceutical compositions described herein include the use of N-oxides, crystalline forms (also known as polymorphs), as well as active metabolites of these lasso peptides having the same type of activity.

[00315] The pharmaceutical compositions described herein can be provided in unit dosage form. A unit dosage form can be a composition containing an amount of a compound that is suitable for administration to a subject (such as a human), in a single dose unit, according to good medical practice. The preparation of a single unit dosage form, however, does not imply that the dosage form is administered once per day or once per course of therapy. Such dosage forms are contemplated to be administered once, twice, thrice or more per day and multiple unit dosage forms can be administered at one time, though a single administration is not specifically excluded. The skilled artisan will recognize that the formulation does not specifically contemplate the entire course of therapy and such decisions are left for those skilled in the art of treatment rather than formulation.

[00316] The amount of the engineered lasso peptide and the additional therapeutic agent (where applicable) can vary depending upon the subject being treated and the particular mode of administration. Particularly, in some embodiments, pharmaceutical compositions provided herein can be formulated so that a dosage of between 0.01-300 mg/kg body weight/day of an engineered lasso peptide can be administered.

7.5 Methods of Using Engineered Lasso Peptides and Pharmaceutical Compositions Thereof

[00317] In another aspect of the present disclosure, provided herein are methods of managing, preventing, and/or treating an endothelin B receptor (ETBR)-mediated proliferative disease in a subject, where the method comprising administering to the subject an effective amount of an engineered lasso peptide or a pharmaceutical composition as described herein. Without being bound by theory, the methods of managing, preventing, and/or treating an endothelin B receptor (ETBR)-mediated proliferative disease in a subject includes turning a cell (e.g;, a tumor cell) from being resistant or less responsive to treatment (e.g., a “cold” tumor) to a cell that is responsive to treatment (e.g., a “hot” tumor), such as an immunotherapy. For example, a cold tumor can be characterized as ETBR being overexpressed on the tumor vasculature, few immune cells in the tumor, and shows resistance to immunotherapy. ETBR overexpression on tumor vasculature shuts down ICAM-1 production, thus diverting immune cells away from a tumor. In contrast, a hot tumor has ETBR inhibited, immune cells infiltrating the tumor, and shows responsiveness to immunotherapy. Inhibiting ETBR allows immune cells to infiltrate tumors and renders them susceptible to immunotherapy. An exemplar}- illustration of such a process is shown in FIG. 6.

[00318] In some embodiments of the present method, the engineered lasso peptide comprises an amino acid sequence as described herein (e.g., Table 1). In some embodiments of the present method, the engineered lasso peptide consists essentially of an amino acid sequence as described herein (e.g., Table 1). In some embodiments of the present method, the engineered lasso peptide consists of an amino acid sequence as described herein (e.g., Table 1) In some embodiments of the present method, the engineered lasso peptide comprises an amino acid sequence as described herein (e.g., Table 1), and possesses the lariat conformation through G1-D9 cyclization. In some embodiments of the present method, the engineered lasso peptide consists essentially of an amino acid sequence as described herein (e.g., Table 1), and possesses the lariat conformation through G1-D9 cyclization. In some embodiments of the present method, the engineered lasso peptide consists of an amino acid sequence as described herein (e.g., Table 1), and possesses the lariat conformation through G1-D9 cyclization.

[00319] In some embodiments, the subject being treated has cells expressing endothelin B receptor (ETBR). In some embodiments, the cells expressing ETBR are endothelial cells in the microenvironment of the neoplastic cells produced by a proliferative disease being treated. In some embodiments, the cells expressing ETBR are endothelial cells of the vasculature in the microenvironment of the neoplastic cells produced by the proliferative disease being treated. In some embodiments, the cells expressing ETBR are neoplastic cells produced by a proliferative disease being treated. In some embodiments, the ETBR expressed by the cells is ETBR1. In some embodiments, the ETBR expressed by the cells is ETBR2. In some embodiments, the ETBR expressed by the cells is ETBR 1 and ETBR2. [00320] In some embodiments, the proliferative disease being treated is cancer. In specific embodiments, the cancer is selected from breast cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), hepatocellular carcinoma, prostate cancer, ovarian cancer, gastric cancer, brain or spinal cancer (e.g., glioma, such as a glioblastoma), melanoma, cancer of the head and neck, colorectal cancer, bladder cancer, vulvar cancer, esophageal squamous cell carcinoma, renal cancer (e.g., clear-cell renal cell carcinoma), cervical cancer, salivary gland carcinoma, lung cancer (e.g., non-small cell lung cancer and small-cell lung cancer), multiple myeloma, or Kaposi’s sarcoma. In particular embodiments, the proliferative disease being treated is melanoma. In particular embodiments, the proliferative disease being treated is breast cancer. In particular embodiments, the proliferative disease being treated is ovarian cancer.

[00321] In specific embodiments, the cancer is melanoma. In specific embodiments, the cancer is esophageal squamous cell carcinoma. In specific embodiments, the cancer is breast cancer. In specific embodiments, the cancer is glioblastoma. In specific embodiments, the cancer is oligodendroglioma. In specific embodiments, the cancer is bladder cancer. In specific embodiments, the cancer is head and neck cancer. In specific embodiments, the cancer is vulvar cancer. In specific embodiments, the cancer is cervical cancer. In specific embodiments, the cancer is ovarian cancer. In specific embodiments, the cancer is prostate cancer. In specific embodiments, the cancer is clear-cell renal cell carcinoma. In specific embodiments, the cancer is multiple myeloma. In specific embodiments, the cancer is pancreatic adenocarcinoma. In specific embodiments, the cancer is pancreatic Kaposi’s sarcoma. In specific embodiments, the cancer is colorectal cancer. In specific embodiments, the cancer is lung cancer.

[00322] In some embodiments, upon administration of the engineered lasso peptide, the maximal percent inhibition of the ETBR-mediated signaling pathway is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, upon administration of the engineered lasso peptide, the maximal percent reduction of ETBR levels is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, upon administration of the engineered lasso peptide, the maximal percent downregulation of ETBR expression is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Accordingly, in some embodiments, the maximal percent inhibition of the ETBR-mediated signaling pathway, the maximal percent reduction of ETBR levels and/or the maximal percent reduction of ETBR levels is at least about 10%. In some embodiments, the maximal percent inhibition of the ETBR-mediated signaling pathway, the maximal percent reduction of ETBR levels and/or the maximal percent reduction of ETBR levels is at least about 20%. In some embodiments, the maximal percent inhibition of the ETBR-mediated signaling pathway, the maximal percent reduction of ETBR levels and/or the maximal percent reduction of ETBR levels is at least about 30%. In some embodiments, the maximal percent inhibition of the ETBR-mediated signaling pathway, the maximal percent reduction of ETBR levels and/or the maximal percent reduction of ETBR levels is at least about 40%. In some embodiments, the maximal percent inhibition of the ETBR-mediated signaling pathway, the maximal percent reduction of ETBR levels and/or the maximal percent reduction of ETBR levels is at least about 50%. In some embodiments, the maximal percent inhibition of the ETBR-mediated signaling pathway, the maximal percent reduction of ETBR levels and/or the maximal percent reduction of ETBR levels is at least about 60%. In some embodiments, the maximal percent inhibition of the ETBR-mediated signaling pathway, the maximal percent reduction of ETBR levels and/or the maximal percent reduction of ETBR levels is at least about 70%. In some embodiments, the maximal percent inhibition of the ETBR-mediated signaling pathway, the maximal percent reduction of ETBR levels and/or the maximal percent reduction of ETBR levels is at least about 80%. In some embodiments, the maximal percent inhibition of the ETBR-mediated signaling pathway, the maximal percent reduction of ETBR levels and/or the maximal percent reduction of ETBR levels is at least about 90%. In some embodiments, the maximal percent inhibition of the ETBR-mediated signaling pathway, the maximal percent reduction of ETBR levels and/or the maximal percent reduction of ETBR levels is at least about 100%.

[00323] In some embodiments, the method includes an engineered lasso peptide that is conjugated to an agent. Such an agent can be selected from the group consisting of a radioisotope, a metal chelator, an enzyme, a protein, a peptide, an antibody, an antibody fragment, a nanobody, a cytotoxic compound, a fluorescent compound, a bioluminescent compound, and a chemiluminescent compound. Accordingly, in some embodiments, the method includes an engineered lasso peptide that is conjugated to a radioisotope. In some embodiments, the method includes an engineered lasso peptide that is conjugated to a metal chelator. In some embodiments, the method includes an engineered lasso peptide that is conjugated to an enzyme. In some embodiments, the method includes an engineered lasso peptide that is conjugated to a protein. In some embodiments, the method includes an engineered lasso peptide that is conjugated to a peptide. In some embodiments, the method includes an engineered lasso peptide that is conjugated to an antibody. In some embodiments, the method includes an engineered lasso peptide that is conjugated to an antibody fragment. In some embodiments, the method includes an engineered lasso peptide that is conjugated to a nanobody. In some embodiments, the method includes an engineered lasso peptide that is conjugated to a cytotoxic compound. In some embodiments, the method includes an engineered lasso peptide that is conjugated to a fluorescent compound. In some embodiments, the method includes an engineered lasso peptide that is conjugated to a bioluminescent compound. In some embodiments, the method includes an engineered lasso peptide that is conjugated to a chemiluminescent compound.

[00324] In some embodiments, the administration of the engineered lasso peptide to the subject inhibits activity of endothelin B receptor (ETBR) expressed by the subject, thereby treating the ETBR-mediated proliferative disease. In some embodiments, the administration of the engineered lasso peptides selectively inhibits ETBR1 over ETBR2. In some embodiments, the administration of the engineered lasso peptides selectively inhibits ETBR2 over ETBR1.

[00325] In some embodiments, administration of the engineered lasso peptide to the subject antagonizes at least one ETBR-mediated signaling pathway. In particular embodiments, the antagonism of the ETBR-mediated signaling pathway is measured by (a) inhibition of release of relaxing factors; (b) upregulation of intercellular adhesion molecule- 1 (ICA.M-1 ) expression and clustering; (c) increasing in migration of intraepitheli al tumor infiltrating leukocytes (TILs) into the microenvironment of the neoplastic cells; (d) inhibition of angiogenesis in the microenvironment of neoplastic cells; (e) inhibition on growth and/or metastasis of neoplastic cells; (f) increasing in apoptosis of neoplastic cells; or any combination of (a) to (f). In particular embodiments, the relaxing factors are selected from nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor, Ca²⁺, protein kinase C, mitogen-activated protein kinase, or any combination thereof. In specific embodiments, the TILs comprises neutrophils, T cells, B cells, NK cells, monocytes or a combination thereof. In specific embodiments, the monocytes comprise macrophages and/or dendritic cells. In some embodiments, the any of the above activities (a) to (f) is inhibited at least 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99% or 100%.

[00326] In some embodiments, administration of the engineered lasso peptide to the subject reduces ETBR levels in the subject, thereby treating the ETBR-mediated proliferative disease. In particular embodiments, administration of the engineered lasso peptide to the subject reduces ETBR levels on the surface of neoplastic cells in the subject, thereby treating the ETBR-mediated proliferative disease. In some embodiments, administration of the engineered lasso peptide to the subject reduces ETBR level in endothelial cells in the microenvironment of the neoplastic cells due to ligand-induced ETBR internalization in the subject, thereby treating the ETBR-mediated proliferative disease. In some embodiments, administration of the engineered lasso peptide to the subject downregulates ETBR expression on the surface of neoplastic cells in the subject, thereby treating the ETBR-mediated proliferative disease. In some embodiments, administration of the engineered lasso peptide to the subject downregulates ETBR expression on the surface of endothelial cells in the microenvironment of the neoplastic cells produced by the proliferative disease in the subject, thereby treating the ETBR-mediated proliferative disease. In some embodiments, administration of the engineered lasso peptides to the subject downregulates ETBR expression in the subject by at least 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99% or 100%.

[00327] In some embodiments, following administration of the engineered lasso peptides to the subject, the inhibition of at least one ETBR-mediated signaling pathway occurs simultaneously as the reduction of ETBR levels as described herein. In some embodiments, the inhibition of at least one ETBR-mediated signaling pathway occurs before the reduction of ETBR levels. In some embodiments, the inhibition of at least one ETBR-mediated signaling pathway occurs after the reduction of ETBR levels. In some embodiments, the reduction of ETBR levels about 1 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 2 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 3 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 4 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 5 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 10 hour after the inhibition of the at least one ETBR- mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 12 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 24 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 36 hour after the inhibition of the at least one ETBR-mediated signaling pathway. In some embodiments, the reduction of ETBR levels occurs about 48 hour after the inhibition of the at least one ETBR-mediated signaling pathway.

[00328] Methods for measuring a reduction of ETBR levels or downregulation of expression of ETBR on the surface of neoplastic cells or levels of endothelial cells in the microenvironment of neoplastic cells are well known in the art, any one of which can be used to measure down regulation.

[00329] In some embodiments, the method further comprises administrating to the subject at least one second therapeutic agent for managing, preventing or treating the proliferative disease, where the second therapeutic agent is not a lasso peptide. In some embodiments, the at least second therapeutic agent is co-administered with one or more of the engineered lasso peptides disclosed herein to the subject either simultaneously or sequentially. In specific embodiments, the second therapeutic agent and the engineered lasso peptide are formulated in a single dosage unit for simultaneous administration. In other embodiments, the second therapeutic agent and the engineered lasso peptide are formulated separately for sequential administration. According to the present disclosure, the at least one second therapeutic agent can be administered before or after administration of the engineered lasso peptides. In specific embodiments where the engineered lasso peptides and the second therapeutic agent are administered sequentially, the time gap between their administration can be at least 1 hour, at least 6 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 1 week, at least 2 weeks, at least 1 months, at least 3 months, at least 6 months or at least 1 year. In some embodiments, the subject has been treated with the second therapeutic agent but is found to be non-responsive to the prior treatment, and the subject is then treated with the engineered lasso peptides of the present disclosure.

[00330] In some embodiments, the engineered lasso peptide provided herein is co- administered with a chemotherapy. In certain embodiments, the chemotherapeutic agent comprises one or more of cyclophosphamide, thiotepa, mechlorethamine (chlormethine/mustine), uramustine, melphalan, chlorambucil, ifosfamide, chlomaphazine, cholophosphamide, estramustine, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, bendamustine, busulfan, improsulfan, piposulfan, carmustine, lomustine, chlorozotocin, fotemustine, nimustine, ranimustine, streptozucin, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, dacarbazine, mitozolomide, temozolomide, paclitaxel, docetaxel, vinblastine, vincristine, vinorelbine, cabazitaxel, dactinomycin (actinomycin D), calicheamicin, dynemicin, amsacrine, doxarubicin, daunorubicin, epirubicin, mitoxantrone, idarubicin, pirarubicin, benzodopa, carboquone, meturedopa, uredopa, altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, callystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancrati statin, sarcodictyin, spongistatin, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomysin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, detorubicin, 6-diazo- 5-oxo-L-norleucine, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, methotrexate, 5- fluorouracil (5-FU), denopterin, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, mitotane, trilostane, frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformithine, elliptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, PSK polysaccharide complex, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2, 2', 2”- trichlorotri ethylamine; T-2 toxin, verracurin A, roridin A and anguidine, urethan, vindesine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (“Ara-C”), etoposide (VP- 16), vinorelbine, novantrone, teniposide, edatrexate, aminopterin, xeloda, ibandronate, irinotecan (e.g., CPT-11), topoisomerase inhibitor RFS 2000, difluorometlhylornithine (DMFO), retinoic acid, capecitabine, plicomycin, gemcitabine, navelbine, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above. In specific embodiments, the chemotherapeutic agent comprises cyclophosphamide.

[00331] In some embodiments, the engineered lasso peptide provided herein is co- administered with an immunotherapy. In certain embodiments, the immunotherapy comprises an immune checkpoint modulator that inhibits, decreases or interferes with the activity of a negative checkpoint regulator. In certain embodiments, the negative checkpoint regulator is selected from Cytotoxic T-lymphocyte antigen-4 (CTLA-4), CD40, CD47, CD80, CD86, Programmed cell death 1 (PD-1), Programmed cell death ligand 1 (PD-L1), Programmed cell death ligand 2 (PD-L2), Lymphocyte activation gene-3 (LAG-3; also known as CD223), Galectin-3, B and T lymphocyte attenuator (BTLA), T-cell membrane protein 3 (TIM3), Galectin-9 (GAL9), B7-H1, B7-H3, B7-H4, T-Cell immunoreceptor with Ig and ITIM domains (TIGIT/Vstm3/WUCAM/VSIG9), V-domain Ig suppressor of T-Cell activation (VISTA), Glucocorticoid-induced tumor necrosis factor receptor-related (GITR) protein, Herpes Virus Entry Mediator (HVEM), 0X40, CD27, CD28, CD137, CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049, CGEN-15052, and CGEN-15092. In certain embodiments, the immune checkpoint inhibitor is an anti -PD-1 antibody. In certain embodiments, the immune checkpoint inhibitor is an anti-PD-Ll antibody.

[00332] In some embodiments, the engineered lasso peptide provided herein is co- administered with a cancer vaccine. In certain embodiments, the cancer vaccine is selected from sipuleucel-T vaccine, Bacillus Calmette-Guerin vaccine, LLO-E7 DNA vaccine, and T- VEC (Imlygic®).

8. SEQUENCES

[00333] The sequences in Tables 2-5 illustrate amino acid and/or nucleic acid sequences that can be used to generate the engineered lasso peptides, recombinant nucleic acids, and/or compositions described herein, and to perform the methods described herein, including those described in the Examples. As needed, an RNA sequence can be readily deduced from the DNA sequence.

Table 2. Listing of amino acid sequences for engineered lasso peptides and encoding nucleotide sequences.

Table 3. Listing of amino acid sequences for precursor engineered lasso peptides and encoding nucleotide sequences.

Table 4. Listing of amino acid sequences for lasso peptidase (B 1 and B2) enzymes, lasso cyclase (C) enzyme, human ETBR, and mouse ETBR.

Table 5. Primer sequences used for Gibson cloning described in Examples 1 and 2.

9. EXAMPLES

[00334] Examples related to the present invention are described below. In most cases, alternative techniques can be used. The examples are intended to be illustrative and are not limiting or restrictive to the scope of the disclose. For example, where engineered lasso peptides are prepared following a protocol of a Scheme, it is understood that conditions may vary, for example, any of the solvents, reaction times, reagents, temperatures, supplements, work up conditions, or other reaction parameters may be varied.

[00335] The following protocols relate to the methods described in Examples 1 to 4.

[00336] General Methods - Methods used for cloning are standard procedures that have described in the literature and are well-known to those experienced in the art. Reagents used for molecular biology experiments were purchased from New England BioLabs (Ipswich, MA), Thermo Fisher Scientific (Waltham, MA), or Gold Biotechnology Inc. (St. Louis, MO). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). The non-methylating Escherichia coli (E. coli donor strain ET12567 carrying the self-transmissible plasmid pUB307 were used for intergeneric transfer of genetic constructs from E. coli to Streptomyces strains. Streptomyces coelicolor M1154, S. coelicolor M1146 and Streptomyces venezuelae ATCC 15439 were used as host strains. All molecular biology manipulations were conducted using standard plates, vials, and flasks typically employed when working with biological molecules such as DNA, RNA and proteins. BQ-788 was purchased from MedChemExpress (Monmouth Junction, NJ). [¹²⁵I]-endothelin-l was purchased from ViTrax Inc. (Placentia, CA). Electrospray ionization mass spectrometry (ESLMS) and tandem mass spectrometry (ESI- MS/MS) analyses were performed using an Agilent 6460C Triple Quadrupole LC/MS system (LC/TQ) equipped with a Jet stream source (AJS), an Agilent 1290 Infinity II LC system, and a diode array detector (DAD). High-resolution LC-MS analyses were performed on an Agilent 6530 Accurate-Mass Q-TOF MS equipped with a dual electrospray ionization source and an Agilent 1260 LC system with diode array detector. MS and UV data were analyzed with Agilent MassHunter Qualitative Analysis version 10.0. Preparative HPLC was carried out using an Agilent 1100 purification system (ChemStation software, Agilent) equipped with an autosampler, multiple wavelength detector, a Prep-LC fraction collector and Phenomenex Luna 5μm Cl 8(2) 150x30 mm preparative column. Semi-preparative HPLC purifications were performed on an Agilent 1100 Series Instrument with a multiple wavelength detector and Phenomenex Luna 5μm C18(2) 250x10 mm semi preparative column. NMR data are acquired using a 600 MHz Bruker Avance III spectrometer with a 1.7 mm cryoprobe. All signals are reported in ppm with the internal DMS0-d6 signal at 2.50 ppm ( ¹H-NMR) or 39.52 ppm (¹³C-NMR). ID data is reported as s = singlet, d = doublet, t = triplet, q=quadruplet, m = multiplet or unresolved, br = broad signal, coupling constant(s) in Hz. [00337] E. coli culturing media - Luria-Bertani (LB) liquid [10 g/L casein peptone, 5 g/L yeast extract, lOg/L NaCl, pH 7.0] and solid [10 g/L casein peptone, 5 g/L yeast extract, 10g/L NaCl, 15 g/L agar, pH 7.0] media were used.

[00338] Streptomyces culturing media - For germination of Streptomyces spore stocks, 2 x YT medium [16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl] was used. For conjugation and selection in Streptomyces strains mannitol soya flour (MS) solid medium [20 g/L mannitol, 20 g/L soya flour, 20 g/L agar] was used. For culturing of Streptomyces strains, maltose yeast extract (MYM) solid medium [4 g/L maltose, 4 g/L yeast extract, 10 g/L malt extract, 20 g/L agar], tryptone soya broth (TSB) medium [17 g/L pancreatic casein peptone, 2.5 g/L K₂HPO₄, 2.5 g/L glucose, 5 g/L NaCl, 3 g/L papain digested soya peptone] and R5 medium [103 g/L sucrose, 0.25 g/L K2SO4, 10.12 g/L MgCl₂.6H₂O, 10 g/L glucose, 0.1 g/L Difco casamino acids, 2 mL/L trace element solution, 5 g/L Difco yeast extract, 5.73 g/L TES buffer. For trace element solution, 40 mg/L ZnCl₂, 200 mg/L FeCl₃.6H₂O, 10 mg/L cuCl₂.2H₂O, 10 mg/L MnCl₂.4H₂O, 10 mg/L Na₂B₄O₇.10H₂O, 10 mg/L (NH₄)₆Mo₇O₂₄.4H₂O; after autoclaving, 10 mL/L KH₂PO₄ (0.5% w/v), 4 mL/L CaCl₂.2H₂O,

15 mL/L L-proline (20% w/v), 7 mL/L NaOH (IN) were used.

The oriT carrying vector plasmid was provided by and purchased from Varigen Biosciences (Middleton, WI) and gBlock fragments were PCR amplified with Q5® High-Fidelity DNA polymerase using primers with the appropriate homologous sequences between neighboring fragments. Vector fragments were DpnI-treated and all PCR fragments were purified with the Zymo DNA Clean and Concentrator® -5 kit. Purified fragments were diluted to 30 fmols and Gibson assembled using NEBuilder® HiFi DNA Assembly Master Mix. After incubation at 50 °C for one hour, 1 μL of the Gibson reaction was electroporated into 50 μL of electrocompetent ET12567 cells.

[00339] Preparation of Electrocompetent ET12567 Cells - a single colony from a freshly streaked plate was used to inoculate a 10 mL LB culture with the antibiotic chloramphenicol (35 μg/mL) in a 50 mL Falcon tube and shaken at 200 rpm at 37 °C. After

16 hours, 500 μL of the overnight culture was used to inoculate a 50 mL LB with chloramphenicol culture in a 250 mL baffled flask, which was shaken at 200 rpm and 37 °C until OD₆₀₀ 0.4. The cultures then were transferred to Falcon tubes on ice and centrifuged for 15 minutes at 4 °C. The supernatant was discarded, and the pellet was washed with 10% glycerol. The wash step was repeated twice and after the last one, the pellet was resuspended in 250 μL 10% glycerol. The cells were aliquoted into Eppendorf tubes, flash frozen and stored at -80 °C.

[00340] Lasso Peptide Isolation - Lasso peptides were extracted from the whole cell broth by first centrifuging the broth in 750 mL Nalgene bottles, then separating the supernatant from the cell pellet. When the lasso peptide was present in both the supernatant and pellet, the pellet was first extracted by addition of a quantity of HPLC grade methanol (MeOH) equal to 3 times the volume of the pellet. This was added directly to the centrifuge bottles, which were shaken overnight on an orbital shaker. The bottles were then centrifuged again, and the methanol extracts collected, pooled, and concentrated on a rotary evaporator to -10-20% methanol in water. This cell extract concentrate was subjected to solid phase extraction (SPE) as follows: 100 g of HP20ss resin (Itochu) was packed into an empty SPE column. The column was washed with 1 L MeOH, then equilibrated with 1 L deionized water. The concentrated extract was loaded onto the SPE resin using a vacuum manifold. The column was washed with 1 L deionized water, then eluted with 1 L 40% MeOH/water, 1 L 50% MeOH/water, 5 x 600 mL 75% MeOH/water, and 1 L 100% MeOH. Each fraction was run on the LC-MS to determine which contained the lasso peptide and approximate the quantity. Fractions containing lasso peptide were pooled and concentrated on a rotary evaporator to remove MeOH, then dried completely on a lyophilizer. Fractions originating from cell pellet material were directly purified on prep HPLC (see below).

Where culture supernatants contain target lasso peptides, the clarified cell broths (via centrifugation at 5,000 x g) were loaded directly to a prepare HP20 column as described above and fractionated as described. Fractions containing target lasso were then dried on a minimal amount of celite, which was loaded into an ISCO Combiflash system using the solid load option. A 50 g HP C18 Redisep column was used for the flash separation. The gradient used DI water as mobile phase A, and HPLC grade methanol as mobile phase B, with the following program: 0 to 20% B over 5 minutes, 20 to 60% B over 25 minutes, 10 minutes at 60% B, 60 to 75% B over 5 minutes, 10 minutes at 65% B, 75 to 100% B over 2 minutes, and 8 minutes at 100% B. Fractions were run on the LCMS quantification method to determine which contained lasso peptide, and those were pooled and concentrated to dryness on a rotary evaporator and lyophilizer. These fractions were then further purified by preparative HPLC (method below). [00341] Lasso Peptide Quantification - The quantity of eluted lasso peptide was determined by

LCMS on an Agilent 6460C Triple Quadrupole LC/MS system (LC/TQ) equipped with a Jet stream source (AJS), an Agilent 1290 Infinity II LC system, and a diode array detector (DAD). UV210 signals corresponding to the lasso peptide were integrated, and area under the curve was used to calculate lasso peptide concentration based on a standard curve generated from a previously generated standard (SEQ ID NO 1). Standard concentrations of SEQ ID NO 1 used were 0.52, 1.3, 3.2, 8, and 20 mg/L. The LCMS method for quantification included the following:

• Column: Phenomenex Kinetex 1.7 μm XB-C18 100 A, 50 x 2.1 mm column.

• Flow rate: 0.4 mL/min

• Temperature: 40 °C

• Mobile Phase A: 0.1% formic acid in water (LCMS grade)

• Mobile Phase B: acetonitrile (LCMS grade)

• Injection amount: 1 μL

• HPLC Gradient: 5% B for 1.0 min, then 5 to 95% B over 3 minutes followed by 95% B for 0.9 min. 1.5 minute post run equilibration time.

[00342] Preparative HPLC - Preparative HPLC was carried out using an Agilent 1100 purification system (ChemStation software, Agilent) equipped with an autosampler, multiple wavelength detector, Prep-LC fraction collector and Phenomenex Luna 5μm C18(2) 150 x 30 mm preparative column. Fractions containing lasso peptides were identified using the LCMS method described above prior to combining and lyophilizing. Product quality control (QC) was performed on the pooled and concentrated lasso fractions (see Product QC method below). The preparative HPLC method included the following:

Column: Phenomenex Luna® preparative column 5 μM, C18(2) 100 A 150 x 30 mm

Flow rate: 20 mL/min

• Temperature: RT

• Mobile Phases: HPLC grade water, MeOH, acetonitrile, isopropyl alcohol, trifluoroacetic acid (TFA), in different percentages and used as gradients

• Injection amount: variable 0.01-2.5 mL

• Example method: Solvent A is water with 0.05% TFA, solvent B is acetonitrile with 0.05% TFA. 35.5% B for 20.0 min, then 35.5 to 95% B over 1 minute followed by 95% B for 3 minutes. 5 minute post run equilibration time.

[00343] Semi-preparative HPLC - If necessary, semi-preparative HPLC purifications were performed on an Agilent 1100 Series Instrument with a multiple wavelength detector. The semipreparative HPLC method included the following:

• Column: Phenomenex Luna 5μm Cl 8(2) 250 x 10 mm

• Flow rate: 4 mL/min

• Temperature: RT

• Mobile Phases: water, MeOH, acetonitrile, trifluoroacetic acid, in different percentages and used as gradients

• Injection amount: variable 0.01 to 1.0 mL

[00344] Sample QC - The purity of eluted lasso peptide was examined by LC-MS on an Agilent 6460C Triple Quadrupole LC/MS system (LC/TQ) equipped with a Jet stream source (AJS), an Agilent 1290 Infinity II LC system, and a diode array detector (DAD). Where possible, MSMS fragmentation was used to further characterize lasso peptides based on the rule described in Fouque, K.J.D, et al., Analyst, 2018,143, 1157-1170, and to confirm amino acid sequences. Proton NMR and high-resolution LCMS data were acquired as described above to further confirm peptide structures. The analytical LCMS method for purity assessment included the following: Column: Phenomenex Kinetex 1.7 μm XB-C18 100 A, 50 x 2.1 mm column.

Flow rate: 0.4 mL/min

• Temperature: 40 °C

• Mobile Phase A: 0.1% formic acid in water (LCMS grade)

• Mobile Phase B: acetonitrile (LCMS grade)

• Injection amount: 1 μL

• HPLC Gradient: 5% B for 1.0 min, then 5 to 50% B over 7 minutes followed by 50 to 95% B over 2 min and 95% B for 2 min. 2 minute post run equilibration time

• After a blank subtraction, the peaks from the TIC, UV210, and UV254 signals were integrated, and purity was reported as the Area Sum % for each signal using Agilent MassHunter Qualitative Analysis 10.0.

[00345] High resolution mass spectrometry - Monoisotopic masses were extrapolated from the lasso peptide charge envelop [(M+H)¹⁺, (M+2H)²⁺, (M+3H)³⁺] in the m/z 500-3,200 range using a Agilent 6530 Accurate-Mass Q-TOF MS equipped with a dual electrospray ionization source and an Agilent 1260 LC system using an internal reference (see analytical procedure described above). Both MS and MS/MS analyses were performed in positive-ion mode.

[00346] Nuclear magnetic resonance (NMR) - NMR samples are dissolved in DMSO-d6 (Cambridge Isotope Laboratories). All NMR experiments are run on a 600 MHz Bruker Avance III spectrometer with a 1.7 mm cryoprobe. All signals are reported in ppm with the internal DMSO-d6 signal at 2.50 ppm (¹H-NMR) or 39.52 ppm (¹³C-NMR). Where applicable, structural characterization of lasso peptide follow the methods described in the following publications:

• Knappe et al., J. Am. Chem. Soc., 2008, 130 (34), 11446-11454

• Maksimov et al., PNAS, 2012, 109 (38), 15223-15228

• Tietz et al., Nature Chem. Bio., 2017,13, 470-478

• Zheng and Price, Prog Nucl Magn Reson Spectrosc, 2010, 56 (3), 267-288

• Marion et al., J Magn Reson, 1989, 85 (2), 393-399

• Davis et al., J Magn Reson, 1991, 94 (3), 637-644

• Rucker and Shaka, Mol Phys, 1989, 68 (2), 509-517

• Hwang and Shaka, J Magn Reson A, 1995, 112 (2), 275-27 Example 1. Cloning, Heterologous Expression, and Production of Parent Lasso Peptide [00347] The following example describes the cloning, heterologous expression, and production of the parent lasso peptide (SEQ ID NO: 1) in two different organisms - treptomyces coelicolor M1154 and M1146 and Streptomyces venezuelae AT CC 15439. The parent lasso peptide produced by the described methods was further analyzed for its inhibition of ETBR using the methods described in Example 3 and for its pharmacokinetic profile using the methods described in Example 4.

Production of Parent Lasso Peptide (SEQ ID NO 1) in Streptomyces coelicolor M1154 and M1146

[00348] Cloning - The entire genomic region consisting of las A. lasB2, lasC, and lasBl genes (SEQ ID NOS: 364, 486, 487, 488; GenBank IDs: WP_106430389.1;

WP_006604202.1; WP_040898778.1; WP_106430390.1;; Table 3) from Streptomyces auratus AGR001 was PCR amplified and cloned into the proprietary pDualP expression vector provided by Varigen Biosciences (FIG. 5), placing the operon expression under the control of the NitR promotor (ε-caprolactam induction, see: Matsumoto, M. et al., Development of nitrilase promoter-derived inducible vectors for Streptomyces (2016). Biosci Biotech Biochem, 80(6), 1230-1237).

[00349] Conjugation - ET12567 competent cells were transformed (electroporation, see general methods) with the lasACB1B2 containing pDualP plasmid and plated in LB agar plates with chloramphenicol (35 μg/mL) and apramycin (50 μg/mL) to select for the incoming plasmid. A single colony was used to inoculate 10 mL LB containing chloramphenicol (35 μg/mL) and apramycin (50 μg/mL) and grown overnight at 37 °C and 200 rpm. A single colony of ET12567/pUB307 was also grown in LB plus chloramphenicol (35 μg/mL) and kanamycin (50 μg/mL) for the triparental mating procedure. The overnight cultures were diluted 1 : 100 in fresh LB plus selective antibiotics and grown at 37 °C until an OD₆₀₀ of 0.4-0.6. The cells were centrifuged at 4000xg washed twice with equal volumes of LB and resuspended in 0.1 volume of LB. On the day of conjugation, 40 μL spores (approximately 10⁶ colony forming units (CFU)) of either S. coelicolor host strains M1154 and M1146, were mixed with 100 μL of 2 x YT medium, heat shocked at 50 °C for 10 minutes and allowed to cool. For each conjugation, 100 μL of the heat shocked spores were mixed with 100 μL of both resuspended E. coli strains. The mixture was plated out on a MS agar plus 20 mM MgCl₂ plate and incubated at 30 °C. After 24 hr the plates were overlayed with 1 mL of filter sterilized molecular biology grade water containing 1 mg of apramycin and 1 mg of nalidixic acid and distributed evenly with a spreader. After 5 to 7 days, potential exconjugant colonies were picked and restreaked onto a MS plate with apramycin (50 μg/mL) and nalidixic acid (25 μg/mL). After additional 3 to 4 days, colony PCR was used on the restreaked colonies to verify that the incoming plasmid had integrated in the host Streptomyces strains.

[00350] Test expression - Three verified colonies from each conjugation were then used to inoculate a 3 mL TSB culture containing apramycin (50 μg/mL) and nalidixic acid (25 μg/mL) in a 15 mL Falcon tube. After 3 days, 400 μL of the TSB culture was used to inoculate a 10 mL R5 culture with apramycin (50 μg/mL) and ε-caprolactam (0.5% w/v) in a 50 mL bio-reaction tube. The same TSB culture was also used to plate a MS plate with apramycin (50 μg/mL) and nalidixic acid (25 μg/mL). R5 cultures were allowed 10 days to grow before checking for production titers.

[00351] Spore stock preparation - The 10-day-old MS plate from the colony with the highest titer was used to make a spore stock. This was done by adding 5 mL of filter sterilized molecular biology grade water to the plate and using a sterile cotton swab to displace the spores, suspending them in water. The spore suspension was transferred into a sterile syringe with a wad of cotton wool at the end and filtered into a 15 mL Falcon tube. The spores were centrifuged at 4000 g for 10 minutes and the supernatant was aspirated with a pipette. The spore pellet was then resuspended in enough 20% glycerol to form an approximately 10% glycerol solution and stored at -80 °C.

[00352] Scale-up production - Starter cultures were prepared by adding 25 μL of the spore stock to 500 μL of 2 x YT medium and heat shocked at 50 °C for 10 minutes. After cooling down, 500 μL of the mixture was used to inoculate 50 mL of TSB containing apramycin (50 μg/mL) in a 250 mL baffled flask. The culture was shaken at 28 °C at 200 rpm for 3 to 4 days, then 20 mL of the starter culture was used to inoculate 500 mL of TSB preculture containing apramycin (50 μg/mL) in a 2 L baffled flask. The culture was shaken at 28 °C at 200 rpm for 2 days. 40 mL of 2-day preculture was used to inoculate 1 L R5 medium containing apramycin (50 μg/mL) and ε-caprolactam (0.5% w/v) in a 2 L baffled flask. The culture was shaken at 28 °C at 200 rpm for 10 days. SEQ ID NO 1 was isolated, purified, and analyzed as described above under the sections Lasso Peptide Isolation and Lasso Peptide Quantification.

Production of Parent Lasso Peptide (SEQ ID NO 1) in Streptomyces venezuelae ATCC 15439 [00353] Cloning - The entire genomic region consisting of lasA. lasB2, lasC, and lasBl genes (SEQ ID NOS: 364, 486, 487, 488; GenBank IDs: WP 106430389.1; WP_006604202.1; WP_040898778.1; WP_106430390.1;; Table 3) from Streptomyces auratus AGR001 was PCR amplified and cloned into the proprietary pDualP expression vector at Varigen Biosciences (FIG. 5), placing the operon expression under the control of the NitR promotor ( ε-caprolactam induction, see: Matsumoto, M. et al., Development of nitrilase promoter-derived inducible vectors for Streptomyces (2016). Biosci Biotech Biochem, 80(6), 1230-1237).

[00354] Conjugation - ET12567 competent cells were transformed (electroporation) with the lasACB1B2 containing pDualP plasmid and plated in LB agar plates with chloramphenicol (35 μg/mL) and apramycin (50 μg/mL) to select for the incoming plasmid. A single colony was used to inoculate 10 mL LB containing chloramphenicol (35 μg/mL) and apramycin (50 μg/mL) and grown overnight at 37 °C and 200 rpm. A single colony of ET12567/pUB307 was also grown in LB plus chloramphenicol (35 μg/mL) and kanamycin (50 μg/mL) for the triparental mating procedure. The overnight cultures were diluted 1 : 100 in fresh LB plus selective antibiotics and grown at 37 °C until an OD₆₀₀ of 0.4-0.6. The cells were centrifuged at 4000 g washed twice with equal volumes of LB and resuspended in 0.1 volume of LB. On the day of conjugation, 15 μL spores (approximately 10⁹ colony forming units (CFU)) of S. venezuelae host strain ATCC 15439 were mixed with 100 μL of 2 x YT medium, heat shocked at 50 °C for 10 minutes and allowed to cool. For each conjugation, 100 μL of the heat shocked spores of the host were mixed with 100 μL of both resuspended E. coll strains. The mixture was plated out on a MS agar plus 20 mM MgCl₂ plate and incubated at 30 °C. After 20 hours, the plates were overlayed with 1 mL of filter sterilized molecular biology grade water containing 1 mg of apramycin and 1 mg of nalidixic acid and distributed evenly with a spreader. After an additional 3 to 4 days, potential exconjugant colonies were picked and restreaked onto a MS plate with apramycin (50 μg/mL) and nalidixic acid (25 μg/mL). After 2 days, colony PCR was used on the restreaked colonies to verify that the incoming plasmid had integrated in the host Streptomyces strain.

[00355] Test production - Three verified colonies from each conjugation were then used to inoculate a 3 mL TSB culture containing apramycin (50 μg/mL) and nalidixic acid (25 μg/mL) in a 15 mL Falcon tube. After 2 days, 400 μL of the TSB culture was used to inoculate a 10 mL R5 culture with apramycin (50 μg/mL) and ε-caprolactam (0.5% w/v) in a 50 mL bio-reaction tube. The same TSB culture was also used to plate a MS plate with apramycin (50 μg/mL) and nalidixic acid (25 μg/mL). R5 cultures were allowed 7 days to grow before checking for production titers. [00356] Spore stock preparation - The 7-day-old MS plate from the colony with the highest titer was used to make a spore stock. This was done by adding 5 mL of filter sterilized molecular biology grade water to the plate and using a sterile cotton swab to displace the spores, suspending them in water. The spore suspension was transferred into a sterile syringe with a wad of cotton wool at the end and filtered into a 15 mL Falcon tube. The spores were centrifuged at 4000 x g for 10 minutes and the supernatant was aspirated with a pipette. The spore pellet was then resuspended in enough 20% glycerol to form an approximately 10% glycerol solution and stored at -80 °C.

[00357] Scale-up production - Starter cultures were prepared by adding 25 μL of the spore stock to 500 μL of 2 x YT medium and heat shocked at 50 °C for 10 minutes. After cooling down, 500 μL of the mixture was used to inoculate 50 mL of TSB containing apramycin (50 μg/mL) in a 250 mL baffled flask. The culture was shaken at 28 °C at 200 rpm for 1-2 days, then 20 mL of the starter was used to inoculate 500 mL of TSB pre-culture containing apramycin (50 μg/mL) in a 2 L baffled flask. The culture was shaken at 28 °C at 200 rpm for 1 day. 40 mL of the 1-day preculture was used to inoculate 1 L of R5 medium containing apramycin (50 μg/mL) and ε-caprolactam (0.5% w/v). The production culture was shaken at 28 °C at 200 rpm for 10 days. SEQ ID NO 1 was isolated, purified, and analyzed as described above under the sections Lasso Peptide Isolation and Lasso Peptide Quantification.

Example 2. Cloning, Heterologous Expression, and Production of Engineered Lassos Peptides

[00358] The following example describes the cloning, heterologous expression, and production of exemplary engineered lasso peptides (SEQ ID NOS: 2-62) in Streptomyces venezuelae ATCC 15439. An exemplary engineered lasso peptide (SEQ ID NO: 35) produced by the described methods was further analyzed for its inhibition of ETBR using the methods described in Example 3 and for its pharmacokinetic profile using the methods described in Example 4.

Production of Engineered Lasso Peptides (SEQ ID NOS: 2-62) in Streptomyces venezuelae ATCC 15439

[00359] Cloning - A 300-base pair (bp) synthetic gene fragment (gBlock) encompassing a region from 138 bp upstream of the gene encoding the engineered lasso peptide (SEQ ID NO: 35) "la A ” (see Table 3 for nucleotide sequence of lasA gene - SEQ ID NO: 398) to 27 bp downstream was PCR amplified and Gibson assembled (see Table 5 and FIG. 5 for primer sequences and location, respectively) with the corresponding fragments of the lasACB1B2 pDualP plasmid described in Example 1. All engineered lasso peptides having SEQ ID NOS: 2-34 and 36-62 were similarly cloned, except that the engineered lasso peptide genes shown as SEQ ID NOs 365-397 and 399-425 in Table 3 were each cloned into a pDualP plasmid containing the mutated lasACB1B2 gene construct. The amino acid positions of SEQ ID NO: 1 that were altered to generate SEQ ID NOS: 2-62 are shown in Table 1 and in FIG. 2. [00360] Conjugation - ET12567 competent cells were transformed (electroporation) with the lasACB1B2 containing pDualP plasmid and plated in LB agar plates with chloramphenicol (35 μg/mL) and apramycin (50 μg/mL) to select for the incoming plasmid. A single colony was used to inoculate 10 mL LB containing chloramphenicol (35 μg/mL) and apramycin (50 μg/mL) and grown overnight at 37 °C and 200 rpm. A single colony of ET12567/pUB307 was also grown in LB plus chloramphenicol (35 μg/mL) and kanamycin (50 μg/mL) for the triparental mating procedure. The overnight cultures were diluted 1 : 100 in fresh LB plus selective antibiotics and grown at 37 °C until an OD₆₀₀ of 0.4-0.6. The cells were centrifuged at 4000 g washed twice with equal volumes of LB and resuspended in 0.1 volume of LB. On the day of conjugation, 15 μL spores (approximately 10⁹ colony forming units (CFU)) of S. venezuelae host strain ATCC 15439 were mixed with 100 μL of 2 x YT medium, heat shocked at 50 °C for 10 minutes and allowed to cool. For each conjugation, 100 μL of the heat shocked spores of the host were mixed with 100 μL of both resuspended E. coll strains. The mixture was plated out on a MS agar plus 20 mM MgCl₂ plate and incubated at 30 °C. After 20 hours, the plates were overlayed with 1 mL of filter sterilized molecular biology grade water containing 1 mg of apramycin and 1 mg of nalidixic acid and distributed evenly with a spreader. After an additional 3 to 4 days, potential exconjugant colonies were picked and restreaked onto a MS plate with apramycin (50 μg/mL) and nalidixic acid (25 μg/mL). After 2 days, colony PCR was used on the restreaked colonies to verify that the incoming plasmid had integrated in the host Streptomyces strain.

[00361] Test production - Three verified colonies from each conjugation were then used to inoculate a 3 mL TSB culture containing apramycin (50 μg/mL) and nalidixic acid (25 μg/mL) in a 15 mL Falcon tube. After 2 days, 400 μL of the TSB culture was used to inoculate a 10 mL R5 culture with apramycin (50 μg/mL) and ε-caprolactam (0.5% w/v) in a 50 mL bio-reaction tube. The same TSB culture was also used to plate a MS plate with apramycin (50 μg/mL) and nalidixic acid (25 μg/mL). R5 cultures were allowed 7 days to grow before checking for production titers. [00362] Spore stock preparation - The 7-day-old MS plate from the colony with the highest titer was used to make a spore stock. This was done by adding 5 mL of filter sterilized molecular biology grade water to the plate and using a sterile cotton swab to displace the spores, suspending them in water. The spore suspension was transferred into a sterile syringe with a wad of cotton wool at the end and filtered into a 15 mL Falcon tube. The spores were centrifuged at 4000 x g for 10 minutes and the supernatant was aspirated with a pipette. The spore pellet was then resuspended in enough 20% glycerol to form an approximately 10% glycerol solution and stored at -80 °C.

[00363] Scale-up production - Starter cultures were prepared by adding 25 μL of the spore stock to 500 μL of 2 x YT medium and heat shocked at 50 °C for 10 minutes. After cooling down, 500 μL of the mixture was used to inoculate 50 mL of TSB containing apramycin (50 μg/mL) in a 250 mL baffled flask. The culture was shaken at 28 °C at 200 rpm for 1-2 days, then 20 mL of the starter was used to inoculate 500 mL of TSB pre-culture containing apramycin (50 μg/mL) in a 2 L baffled flask. The culture was shaken at 28 °C at 200 rpm for 1 day. 40 mL of the 1-day preculture was used to inoculate 1 L of R5 medium containing apramycin (50 μg/mL) and ε-caprolactam (0.5% w/v). The production culture was shaken at 28 °C at 200 rpm for 10 days. SEQ ID NO 35 was isolated, purified, and analyzed as described above under the sections Lasso Peptide Isolation and Lasso Peptide Quantification.

Example 3. Inhibition of ETBR Expressed on the Surface of CHO Cells

[00364] Cell culture CHO cells were maintained in Kaighn’s F-12K medium supplemented with 10% FB Essence and 2 mM glutamate under a humidified 5% CO2-95% air atmosphere.

[00365] Transient expression of ETBR in CHO cells. Cell lines transiently expressing ETBR (CHO-ETBR) are obtained using a mammalian HA-epitope tag expression vector, pHM6 (Roche Applied Science), that carries a cDNA construct encoding human recombinant ETBR receptor (SEQ ID NO 484; Table 4; GenBank Accession Number NP 000106) or mouse recombinant ETBR receptor (SEQ ID NO 485; Table 4; GenBank Accession Number NP 031930.1). Each expression vector was introduced into CHO cells by lipofection using Lipofectamine 2000 (Thermo Fisher, Carlsbad, CA, USA) according to the manufacturer’s instructions. Confirmation of ETBR gene expression was confirmed in cell populations by surface staining with antibodies (anti-HA tag AlexaFluor 488 conjugated mouse IgG, R&D Systems, cat # IC6875G) in combination with flow cytometry. Binding experiments are conducted with membranes prepared from the transiently transfected CHO-ETBR cells.

[00366] Preparation of Plasma Membranes for Radioligand Binding Studies. CHO- K1 cells expressing recombinant ETBR receptors were cultured under standard conditions at 37°C/5% CO2. Cells were collected in ice-cold phosphate buffered saline, pH 7.4 (PBS), and subsequently centrifuged at 500 x g for 5 min at 4°C. The resulting cell pellet was then resuspended in cell lysis buffer containing 5 mM HEPES, pH 7.4 containing 10 mM EDTA and 2 mM EGTA, homogenized on ice by Dounce homogenization, and centrifuged (48,000 x g for 15 min at 4°C). The initial pellet was washed twice more by resuspending in 20 mM HEPES, pH 7.4, on ice, and centrifuged as before (48,000 x g for 15 min at 4°C). Crude membrane pellets were aliquoted and stored at -80°C prior to use in radioligand binding assays.

Radioligand Binding Assay Protocols.

[00367] For competition binding studies. The total assay volume in each well was 200 μL and used 96-well microwell plates. Reagent volumes consisted of 3 μL/well of DMSO containing various lasso peptides (for example, having amino acid sequences described in Table 1) prepared at a range of concentrations, 50 μL/well of [¹²⁵I]-endothelin-l diluted in Assay Buffer (20 mM HEPES, 10 mM MgCl₂, 0.2% bovine serum albumin (BSA), pH 7.4), and 150 ul/well of diluted ETAR or ETBR expressing membranes prepared in Assay Buffer. All reagents were combined and incubated for 2 hours at room temperature. Assay incubations were terminated by rapid filtration through Perkin Elmer GF/C filtration plates under vacuum pressure using a 96-well Packard filtration apparatus, followed by washing the filter plates five times with ice-cold Assay Buffer. Plates were then dried at 45°C for a minimum of four hours. Finally, 25 μL of BetaScint scintillation cocktail was added to each well and the plates were counted in a Packard TopCount NXT scintillation counter.

[00368] Total and non-specific binding were measured in the presence and absence of 10 μM of known endothelin B receptor antagonist BQ-788 (Ishikawa, K. et al., Biochemical and pharmacological profile of a potent and selective endothelin B-receptor antagonist, BQ-788 (1994). Proc. Natl. Acad. Sci. USA, 91, 4892-4896). Non-linear regression was used for analysis of competitive inhibition curves of lasso peptides and experimentally determined IC₅₀ values were used to calculate the dissociation constant (Ki) for each compound using the Cheng-Prusoff equation. ETBR binding data for engineered lasso peptide analogs is shown in Table 6. Additionally, exemplary binding curves for the parent lasso peptide and several exemplary engineered lasso peptides are shown in FIGS. 7-13. Table 6. Human and Mouse ETBR Binding Results for Parent Lasso Peptide (SEQ ID NO 1) and Exemplary Engineered Lasso Peptides

ND = not detected

[00369] Saturation binding studies for determination of radioligand affinity constant (Kd). Firstly, 3 μL/well of either DMSO or DMSO containing BQ-788 at a final concentration of 10 μM were added to define total and non-specific binding respectively. Secondly, 50 μL/well of assay buffer with serial diluted [¹²⁵I]-endothelin-l was added. The final concentration of radioligand ranged from 0.015 to 5 nM, calculated based on the stock radioactivity concentration and the specific activity (2200Ci/mmol). Thirdly, 10 ug/well of diluted membranes were added to initiate the assay. Quadruplicate wells were used for each concentration in the assay. Wells were incubated for 2 hours at room temperature. Assay incubations were terminated by rapid filtration through Perkin Elmer GF/C filtration plates under vacuum pressure using a 96-well Packard filtration apparatus, as described above. The dissociation constant (Kd) of [¹²⁵I]-endothelin-1 was calculated using non-linear regression analysis of the specific amount of radioactivity bound to the membrane as a function of the radioligand concentration.

Example 4. Pharmacokinetic Study of the Parent Lasso Peptide and an Exemplary Engineered Lasso Peptide in CD-I mice

[00370] This example shows the pharmacokinetic assessment of the parent lasso peptide (SEQ ID NO 1) and an exemplary engineered lasso peptide (SEQ ID NO: 35).

[00371] Pharmacokinetic (PK) assessments of the parent lasso peptide (SEQ ID NO 1) were performed as follows. Male CD-1 mice, weighing 30 - 42 g, were obtained from Charles River and acclimated for at least 3 days. All mice were housed in separate cages and given ad libitum access to water and food pellets. The doses and routes of administrations are listed in Table 7. On the day of dosing, all mice were bled according to the PK timepoints outlined in the Table 8 below, depending on the route of administration. All timepoints, except for the last timepoint were bled via the submandibular vein, and the terminal bleed was via the vena cava. Approximately 0.2 mL of whole blood is collected per each timepoint. Data was analyzed according to the bioanalytical method summarized in Table 9. All experimental procedures were applied in strict accordance with IACUC guidelines. Pharmacokinetic data for SEQ ID NO 1 is shown in Table 10.

[00372] Animals were administered SEQ ID NO 1, as described in Table 7 below.

Table 7. Group Designation and Dosage Levels *

* Vehicle is 10% DMSO, 30% PEG400, 60% water

** Route of Administration. IV: single injection into tail vein; IP: single injection through peritoneum and into the abdominal cavity. Table 8. Blood Sample Collection, Processing, Storage, and Shipment

Collection Intervals: One collection day

Animals/Time Point: Group 1:

Group 2:

Collection Volume: Approximately 0.2 mL of whole blood per time point

Collection Site and Method: Submandibular (non-terminal bleed) or vena cava

(terminal bleed)

Anticoagulant: K₂EDTA

Sample Requirements: Samples will be collected in K₂EDTA tubes and centrifuged under refrigerated conditions to collect plasma. The plasma will be frozen at -70°C until transferred for bioanalysis.

[00373] The non-validated bioanalytical method that was used for the detection of SEQ ID NO 1 in mouse plasma is summarized in Table 9.

Table 9. Bioanalytical Method Summary for SEQ ID NO 1

[00374] The results from the above study of SEQ ID NO 1 is presented in Table 10.

Table 10. PK Parameters for SEQ ID NO 1

[00375] Similarly, SEQ ID NO 35 was examined for PK parameters in the same fashion.

The results of this analysis is presented in Table 11.

Table 11. PK Parameters for SEQ ID NO 35

iv sampling timepoints = 5 min, 30 min, 1 h, 2 h, 4 h, 24 h; male CD1 mice; * Parameter range is reflective of multi-phasic compartment model

Claims

CLAIMS What is claimed is:

1. An engineered lasso peptide comprising a variant of amino acid sequence SEQ ID NO: 1, wherein the engineered lasso peptide comprises one or more amino acid substitutions, and wherein the engineered lasso peptide, when cyclized, has at least a 1.5-fold higher specific binding affinity to endothelin B receptor (ETBR) compared to a lasso peptide consisting of the amino acid sequence of SEQ ID NO: 1.

2. The engineered lasso peptide of claim 1, wherein the one or more amino acid substitutions allows for increased hydrogen bonding of the engineered lasso peptide to residues in the pocket of ETBR that bind endothelin ligands or in the receptor capping region of ETBR.

3. The engineered lasso peptide of claim 1, wherein the one or more amino acid substitutions comprises aF11Y substitution.

4. The engineered lasso peptide of claim 3, wherein the one or more amino acid substitutions further comprise a substitution selected from the group consisting of N2A, N2D, N2S, W3A, W3D, W3E, W3Y, W3H, H4A, H4I, H4Q, H4M, H4L, H4W, H4Y, G5A, T6A, T6E, T6I, T6K, T6L, T6V, T6S, T6H, S7E, S7F, S7I, S7L, S7N, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, W10A, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, W16E, and W16K.

5. The engineered lasso peptide of claim 3, wherein the one or more amino acid substitutions further comprise one amino acid substitution selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, and W16K.

6. The engineered lasso peptide of claim 1, wherein the one or more amino acid substitutions comprises a F12H substitution.

7. The engineered lasso peptide of claim 6, wherein the one or more amino acid substitutions further comprise a substitution selected from the group consisting of N2A, N2D, N2S, W3A, W3D, W3E, W3Y, W3H, H4A, H4I, H4Q, H4M, H4L, H4W, H4Y, G5A, T6A, T6E, T6I, T6K, T6L, T6V, T6S, T6H, S7E, S7F, S7I, S7L, S7N, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, W10A, F11Y, F11S, F11T, F11W, N13S, N13F, N13H, Y15F, Y15L, Y15H, W16E, and W16K. The engineered lasso peptide of claim 6, wherein the one or more amino acid substitutions further comprise one amino acid substitution selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F11Y, F11 S, F11T, N13F, N13H, Y15F, Y15L, Y15H, and W16K. The engineered lasso peptide of claim 1, wherein the one or more amino acid substitutions comprises a F12Y substitution. The engineered lasso peptide of claim 9, wherein the one or more amino acid substitutions further comprise a substitution selected from the group consisting of N2A, N2D, N2S, W3A, W3D, W3E, W3Y, W3H, H4A, H4I, H4Q, H4M, H4L, H4W, H4Y, G5A, T6A, T6E, T6I, T6K, T6L, T6V, T6S, T6H, S7E, S7F, S7I, S7L, S7N, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, W10A, F11Y, F11 S, F11T, F11W, N13S, N13F, N13H, Y15F, Y15L, Y15H, W16E, and W16K. The engineered lasso peptide of claim 9, wherein the one or more amino acid substitutions further comprise one amino acid substitution selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F11Y, F11 S, F11T, N13F, N13H, Y15F, Y15L, Y15H, and W16K. The engineered lasso peptide of claim 3, wherein the one or more amino acid substitutions are selected from the group consisting of: a) H4L andF11Y; b) H4M andF11Y; c) F11Y and F12H; and d) F11Y and F12Y. The engineered lasso peptide of claim 3, wherein the one or more amino acid substitutions further comprise two amino acid substitutions selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, and W16K. The engineered lasso peptide of claim 13, wherein the one or more amino acid substitutions are selected from the group consisting of: a) H4M, F11Y, and F12Y; b) W3E, H4M, andF11Y; c) W3 Y, H4M, and F 11 Y; and d) W3H, H4M, andF11Y. The engineered lasso peptide of claim 3, wherein the one or more amino acid substitutions further comprise three amino acid substitutions selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, and W16K. The engineered lasso peptide of claim 15, wherein the one or more amino acid substitutions are selected from the group consisting of: a) W3D, H4M, F11Y, and F12Y; b) W3E, H4M, F11Y, and F12Y; c) W3Y, H4M, F11Y, and F12Y; and d) W3H, H4M, F11Y, and F12Y. The engineered lasso peptide of claim 3, wherein the one or more amino acid substitutions further comprise four amino acid substitutions selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, and W16K. The engineered lasso peptide of claim 17, wherein the one or more amino acid substitutions are selected from the group consisting of: a) W3D, H4M, F11Y, F12Y, and N13F; b) W3E, H4M, F11Y, F12Y, and N13F; c) W3Y, H4M, F11Y, F12Y, and N13F; d) W3H, H4M, F11Y, F12Y, and N13F; e) W3D, H4M, F11Y, F12Y, and N13H; f) W3E, H4M, F11Y, F12Y, and N13H; g) W3Y, H4M, F11Y, F12Y, and N13H; and h) W3H, H4M, F11Y, F12Y, and N13H. The engineered lasso peptide of claim 3, wherein the one or more amino acid substitutions further comprise five amino acid substitutions selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, and W16K. The engineered lasso peptide of claim 19, wherein the one or more amino acid substitutions are selected from the group consisting of: a) W3D, H4M, F11Y, F12Y, N13F, and S7I; b) W3E, H4M, F11Y, F12Y, N13F, and S7I; c) W3Y, H4M, F11Y, F12Y, N13F, and S7I; d) W3H, H4M, F11Y, F12Y, N13F, and S7I; e) W3D, H4M, F11Y, F12Y, N13H, and S7I; f) W3E, H4M, F11Y, F12Y, N13H, and S7I; g) W3Y, H4M, F11Y, F12Y, N13H, and S7I; h) W3H, H4M, F11Y, F12Y, N13H, and S7I; i) W3D, H4M, F11Y, F12Y, N13F, and S7Y; j) W3E, H4M, F11Y, F12Y, N13F, and S7Y; k) W3Y, H4M, F11Y, F12Y, N13F, and S7Y; l) W3H, H4M, F11Y, F12Y, N13F, and S7Y; m) W3D, H4M, F11Y, F12Y, N13H, and S7Y; n) W3E, H4M, F11Y, F12Y, N13H, and S7Y; o) W3Y, H4M, F11Y, F12Y, N13H, and S7Y; p) W3H, H4M, F11Y, F12Y, N13H, and S7Y; q) W3D, H4M, F11Y, F12Y, N13F, and S7F; r) W3E, H4M, F11Y, F12Y, N13F, and S7F; s) W3Y, H4M, F11Y, F12Y, N13F, and S7F; t) W3H, H4M, F11Y, F12Y, N13F, and S7F; u) W3D, H4M, F11Y, F12Y, N13H, and S7F; v) W3E, H4M, F11Y, F12Y, N13H, and S7F; w) W3Y, H4M, F11Y, F12Y, N13H, and S7F; x) W3H, H4M, F11Y, F12Y, N13H, and S7F; y) W3D, H4M, F11Y, F12Y, N13F, and S7K; z) W3E, H4M, F11Y, F12Y, N13F, and S7K; aa) W3Y, H4M, F11Y, F12Y, N13F, and S7K; bb) W3H, H4M, F11Y, F12Y, N13F, and S7K; cc) W3D, H4M, F11Y, F12Y, N13H, and S7K; dd) W3E, H4M, F11Y, F12Y, N13H, and S7K; ee) W3Y, H4M, F11Y, F12Y, N13H, and S7K; ff) W3H, H4M, F11Y, F12Y, N13H, and S7K; gg) W3D, H4M, F11Y, F12Y, N13F, and S7R; hh) W3E, H4M, F11Y, F12Y, N13F, and S7R; ii) W3Y, H4M, F11Y, F12Y, N13F, and S7R; jj) W3H, H4M, F11Y, F12Y, N13F, and S7R; kk) W3D, H4M, F11Y, F12Y, N13H, and S7R;

11) W3E, H4M, F11Y, F12Y, N13H, and S7R; mm) W3Y, H4M, F11Y, F12Y, N13H, and S7R; and nn) W3H, H4M, F11Y, F12Y, N13H, and S7R. An engineered lasso peptide comprising a variant of amino acid sequence SEQ ID NO: 1, wherein the engineered lasso peptide comprises one or more amino acid substitutions selected from the group consisting of N2A, W3A, W3D, W3E, W3Y, W3H, H4A, H4Q, H4M, H4Y, G5A, T6A, T6V, T6H, S7F, S7I, S7L, S7W, S7Y, S7K, S7R, S7P, P8F, P8L, F11Y, F11 S, F11T, F12H, F12L, F12M, F12W, F12Y, N13S, N13F, N13H, Y15F, Y15L, Y15H, and W16K. The engineered lasso peptide of claim 21, wherein the one or more amino acid substitutions comprises two, three, four, five, or six amino acid substitutions. The engineered lasso peptide of claim 21, wherein the one or more amino acid substitutions comprises two amino acid substitutions. The engineered lasso peptide of claim 23, wherein the two amino acid substitutions are selected from the group consisting of: a) H4L andF11Y; b) H4M andF11Y; c) T6P and P8F; d) T6P and P8L; e) T6V and S7N; f) S7P and P8F; g) S7P and P8L; h) F11Y and F12H; and i) F11Y and F12Y. The engineered lasso peptide of claim 21, wherein the one or more amino acid substitutions comprises three amino acid substitutions. The engineered lasso peptide of claim 25, wherein the three amino acid substitutions are selected from the group consisting of: a) H4M, F11Y, and F12Y; b) W3E, H4M, andF11Y; c) W3 Y, H4M, and F 11 Y; and d) W3H, H4M, andF11Y. The engineered lasso peptide of claim 21, wherein the one or more amino acid substitutions comprises four amino acid substitutions. The engineered lasso peptide of claim 27, wherein the four amino acid substitutions are selected from the group consisting of: a) W3D, H4M, F11Y, and F12Y; b) W3E, H4M, F11Y, and F12Y; c) W3Y, H4M, F11Y, and F12Y; and d) W3H, H4M, F11Y, and F12Y. The engineered lasso peptide of claim 21, wherein the one or more amino acid substitutions comprises five amino acid substitutions. The engineered lasso peptide of claim 29, wherein the five amino acid substitutions are selected from the group consisting of: a) W3D, H4M, F11Y, F12Y, and N13F; b) W3E, H4M, F11Y, F12Y, and N13F; c) W3Y, H4M, F11Y, F12Y, and N13F; d) W3H, H4M, F11Y, F12Y, and N13F; e) W3D, H4M, F11Y, F12Y, and N13H; f) W3E, H4M, F11Y, F12Y, and N13H; g) W3Y, H4M, F11Y, F12Y, and N13H; and h) W3H, H4M, F11Y, F12Y, and N13H. The engineered lasso peptide of claim 21, wherein the one or more amino acid substitutions comprises six amino acid substitutions. The engineered lasso peptide of claim 32, wherein the six amino acid substitutions are selected from the group consisting of: a) W3D, H4M, F11Y, F12Y, N13F, and S7I; b) W3E, H4M, F11Y, F12Y, N13F, and S7I; c) W3Y, H4M, F11Y, F12Y, N13F, and S7I; d) W3H, H4M, F11Y, F12Y, N13F, and S7I; e) W3D, H4M, F11Y, F12Y, N13H, and S7I; f) W3E, H4M, F11Y, F12Y, N13H, and S7I; g) W3Y, H4M, F11Y, F12Y, N13H, and S7I; h) W3H, H4M, F11Y, F12Y, N13H, and S7I; i) W3D, H4M, F11Y, F12Y, N13F, and S7Y; j) W3E, H4M, F11Y, F12Y, N13F, and S7Y; k) W3Y, H4M, F11Y, F12Y, N13F, and S7Y; l) W3H, H4M, F11Y, F12Y, N13F, and S7Y; m) W3D, H4M, F11Y, F12Y, N13H, and S7Y; n) W3E, H4M, F11Y, F12Y, N13H, and S7Y; o) W3Y, H4M, F11Y, F12Y, N13H, and S7Y; p) W3H, H4M, F11Y, F12Y, N13H, and S7Y; q) W3D, H4M, F11Y, F12Y, N13F, and S7F; r) W3E, H4M, F11Y, F12Y, N13F, and S7F; s) W3Y, H4M, F11Y, F12Y, N13F, and S7F; t) W3H, H4M, F11Y, F12Y, N13F, and S7F; u) W3D, H4M, F11Y, F12Y, N13H, and S7F; v) W3E, H4M, F11Y, F12Y, N13H, and S7F; w) W3Y, H4M, F11Y, F12Y, N13H, and S7F; x) W3H, H4M, F11Y, F12Y, N13H, and S7F; y) W3D, H4M, F11Y, F12Y, N13F, and S7K; z) W3E, H4M, F11Y, F12Y, N13F, and S7K; aa) W3Y, H4M, F11Y, F12Y, N13F, and S7K; bb) W3H, H4M, F11Y, F12Y, N13F, and S7K; cc) W3D, H4M, F11Y, F12Y, N13H, and S7K; dd) W3E, H4M, F11Y, F12Y, N13H, and S7K; ee) W3Y, H4M, F11Y, F12Y, N13H, and S7K; ff) W3H, H4M, F11Y, F12Y, N13H, and S7K; gg) W3D, H4M, F11Y, F12Y, N13F, and S7R; hh) W3E, H4M, F11Y, F12Y, N13F, and S7R; ii) W3Y, H4M, F11Y, F12Y, N13F, and S7R; jj) W3H, H4M, F11Y, F12Y, N13F, and S7R; kk) W3D, H4M, F11Y, F12Y, N13H, and S7R;

11) W3E, H4M, F11Y, F12Y, N13H, and S7R; mm) W3Y, H4M, F11Y, F12Y, N13H, and S7R; and nn) W3H, H4M, F11Y, F12Y, N13H, and S7R. The engineered lasso peptide of any one of claims 1 to 32, wherein the engineered lasso peptide is at least 10% more stable as measured by thermal degradation or proteolytic degradation through hydrolysis of a peptide bond compared to a lasso peptide consisting of the amino acid sequence of SEQ ID NO: 1. The engineered lasso peptide of any one of claims 1 to 32, wherein the engineered lasso peptide is at least 10% more soluble in water or a mixture containing water compared to a lasso peptide consisting of the amino acid sequence of SEQ ID NO: 1. An engineered lasso peptide comprising an amino acid sequence selected from SEQ ID NOS: 2-117. An engineered lasso peptide consisting of an amino acid sequence selected from SEQ ID NOS: 2-117. The engineered lasso peptide of any one of claims 1 to 35, wherein the engineered lasso peptide further comprises a leader sequence. The engineered lasso peptide of claim 37, wherein the leader sequence comprises the amino acid sequence of MTEQSEQTPTEYIPPMLVEVGEFTEDTL (SEQ ID NO: 118). The engineered lasso peptide of any one of claims 1 to 35, wherein the engineered lasso peptide further comprises a C-terminal tryptophan (W) modification selected from the group consisting of: a) tryptophan having a C-terminal methyl ester group (-CO2Me) in place of the carboxylic acid group (-CO2H) (W-OMe); b) tryptophan having a C-terminal benzyl ester group (-CO2Bn) in place of the carboxylic acid group (-CO2H) (W-OBn); c) tryptophan having a C-terminal amide group (-CONH2) in place of the carboxylic acid group (-CO2H) (W-NH2); d) 7-hydroxyl-trptophan (W-7-OH); e) 2-naphthylalanine (Nal) in place of W; and f) an aza derivative of tryptophan - (2S)-2-amino-3-(lH-pyrrolo[5,4-b]pyridin-3- yl)propanoic acid - in place of W having the structure of:

(Tm). The engineered lasso peptide of any one of claims 1 to 36 and 39, wherein the engineered lasso peptide is G1-D9 cyclized. The engineered lasso peptide of claim 40, wherein the engineered lasso peptide competes with endothelin for the binding with ETBR. The engineered lasso peptide of claim 41, wherein the endothelin is endothelin 1, endothelin 2 and/or endothelin 3. The engineered lasso peptide of claim 40, wherein the engineered lasso peptide preferentially binds to ETBR over endothelin A receptor (ETAR). The engineered lasso peptide of claim 40, wherein the engineered lasso peptide specifically antagonizes ETBR. The engineered lasso peptide of claim 40, wherein the engineered lasso peptide preferentially binds to ETBR1 over ETBR2. The engineered lasso peptide of claim 45, wherein the engineered lasso peptide specifically antagonizes ETBR1. A pharmaceutical composition comprising the engineered lasso peptide of any one of claims 1 to 46 and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition of claim 47, wherein the composition further comprises a second therapeutic agent for managing, preventing or treating cancer. The pharmaceutical composition of claim 48, wherein the second therapeutic agent is a chemotherapy or immunotherapy for cancer. The pharmaceutical composition of claim 49, wherein the immunotherapy is an anti- cancer vaccine or immune checkpoint modulator. A method of managing, preventing, or treating an endothelin B receptor (ETBR)- mediated proliferative disease producing neoplastic cells in a subject, comprising administering to the subject a therapeutically effective amount of an engineered lasso peptide of any one of claims 1 to 46 or the pharmaceutical composition of any one of claim 47 to 50. The method of claim 51, wherein upon administration, the engineered lasso peptide a) antagonizes an ETBR-mediated signaling pathway; b) reduces ETBR levels on the surface of neoplastic cells and/or endothelial cells in the microenvironment of the neoplastic cells due to ligand-induced ETBR internalization; and/or c) downregulates ETBR expression on the surface of the neoplastic cells and/or endothelial cells in the microenvironment of the neoplastic cells. The method of claim 52, wherein antagonism of the ETBR-mediated signaling pathway is measured by: a) inhibition of release of relaxing factors; b) upregulation of intercellular adhesion molecule- 1 (ICAM-1) expression and clustering; c) increase in migration of intraepithelial tumor infiltrating leukocytes (TILs) into the microenvironment of the neoplastic cells; d) inhibition of angiogenesis in the microenvironment of neoplastic cells; e) inhibition of growth and/or metastasis of neoplastic cells; and/or f) increase in apoptosis of neoplastic cells. The method of claim 53, wherein the relaxing factors are nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor, Ca²⁺, protein kinase C, mitogen- activated protein kinase, or any combination thereof. The method of claim 53, wherein the TILs comprise neutrophils, T cells, B cells, NK cells, monocytes or a combination thereof. The method of claim 55, wherein the monocytes comprise macrophages and/or dendritic cells. The method of any one of claims 51 to 56, wherein the neoplastic cells express ETBR. The method of any one of claims 51 to 57, wherein the subject expresses ETBR in endothelial cells of the vasculature in the microenvironment of the neoplastic cells. The method of claim 57 or 58, wherein the ETBR is ETBR1 and/or ETBR2 The method of any one of claims 51 to 59, wherein proliferative disease is cancer. The method of claim 60, wherein the cancer is breast cancer, pancreatic cancer, hepatocellular cancer, prostate cancer, ovarian cancer, gastric cancer, brain or spinal cancer, melanoma, cancer of the head and neck, colorectal cancer, bladder cancer, vulvar cancer, esophageal squamous cell carcinoma, renal cancer, cervical cancer, salivary gland carcinoma, lung cancer, multiple myeloma, or Kaposi’s sarcoma. The method of claim 61, wherein the brain or spinal cancer is a glioma. The method of claim 62, wherein the glioma is a glioblastoma. The method of claim 61, wherein the cancer is melanoma, breast cancer, or ovarian cancer. The method of claim 52, wherein antagonism of the ETBR-mediated pathway is a maximal percent inhibition of at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. The method of claim 52, wherein the reduction of ETBR levels is a maximal percent reduction of at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. The method of claim 52, wherein the downregulation of ETBR expression is a maximal percent downregulation of at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. The method of any one of claims 51 to 67, wherein the engineered lasso peptide is conjugated to an agent. The method of claim 68, wherein the agent is selected from the group consisting of a radioisotope, a metal chelator, an enzyme, a protein, a peptide, an antibody, an antibody, an antibody fragment, a nanobody, a cytotoxic compound, a fluorescent compound, a bioluminescent compound, and a chemiluminescent compound. The method of any one of claims 51 to 69, further comprising co-administering to the subject a second therapeutic agent with the engineered lasso peptide. The method of claim 70, wherein the second therapeutic agent is conjugated with the engineered lasso peptide. The method of claim 70 or 71, wherein the second therapeutic agent is an immunotherapy or chemotherapy. The method of claim 71, wherein the immunotherapy is an anti-cancer vaccine or an immune checkpoint modulator. A recombinant nucleic acid encoding the engineered lasso peptide of any one of claims 1 to 37. The recombinant nucleic acid of claim 74, wherein the recombinant nucleic acid comprises a nucleotide sequence selected from SEQ ID NO: 119-235 encoding the engineered lasso peptide. A recombinant nucleic acid encoding a lasso precursor peptide comprising an amino acid sequence selected from SEQ ID NOS: 238-363. The recombinant nucleic acid of claim 76, wherein the recombinant nucleic acid comprises a nucleotide sequence selected from SEQ ID NOS: 364-480 encoding the lasso precursor peptide. The recombinant nucleic acid of any one of claims 74 to 77, wherein the recombinant nucleic acid comprises a nucleotide sequence encoding the engineered lasso peptide operatively linked to a promoter. A vector comprising the recombinant nucleic acid of any one of claims 74 to 78. A non-naturally occurring microbial organism comprising the recombinant nucleic acid of any one of claims 74 to 78 or the vector of claim 79. A method for producing an engineered lasso peptide using a non-naturally occurring microbial organism, wherein the method comprises: a) introducing into the microbial organism a first nucleic acid comprising the recombinant nucleic acid of any one of claims 74 to 78 or the vector of claim 79 and a second nucleic acid encoding a lasso peptide biosynthesis component; and b) culturing the microbial organism under a condition suitable for lasso formation to produce the engineered lasso peptide. The method of claim 81, wherein the first nucleic acid encodes the engineered lasso peptide of claim 37, and wherein the lasso peptide biosynthesis component comprises a lasso peptidase capable of catalyzing removal of the leader sequence. The method of claim 81 or 82, wherein the lasso peptide biosynthesis component comprises a lasso cyclase capable of cyclizing a linear lasso core sequence to a mature lasso peptide. The method of claim 81 or 82, wherein the lasso peptide biosynthesis component comprises a lasso peptidase and a lasso cyclase, and wherein the method comprises introducing the second nucleic acid sequence encoding the lasso cyclase and a third nucleic acid sequence encoding the lasso peptidase. The method of claim 81 or 82, wherein the lasso peptide biosynthesis component comprises a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE). The method of claim 81 or 82, wherein the lasso peptide biosynthesis component comprises a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE), and wherein the method comprises introducing the second nucleic acid sequence encoding the lasso cyclase and a fourth nucleic acid sequence encoding the RRE. The method of claim 81 or 82, wherein the lasso peptide biosynthesis component comprises a lasso peptidase, a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE). The method of claim 81 or 82, wherein the lasso peptide biosynthesis component comprises a lasso peptidase, a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE), and wherein the method comprises introducing the second nucleic acid sequence encoding the lasso cyclase, a third nucleic acid sequence encoding the lasso peptidase, and a fourth nucleic acid sequence encoding the RRE. The method of any one of claims 81 to 88, wherein at least two of the first, second, third and fourth nucleic acid sequences are in a same nucleic acid molecule. The method of any one of claims 81 to 89, wherein the microbial organism is E.coli, Vibrio natriegens, Burholderia spp., Corynebacterium glutamicum, or Sphingomonas subterranean, Pseudomonas fluorescens, Saccharomyces cerevisiae, Pichia pasloris, Rhodococcus jostii, Saccharopolyspora erylhraea, Streptomyces lividans, Streptomyces coelicolor, Streptomyces albus, or Streptomyces venezuelae . The method of any one of claims 81 to 90, wherein the culturing is performed under aerobic and/or glucose-limiting conditions. The method of any one of claims 81 to 91, wherein the method further comprises isolating the engineered lasso peptide from the culture medium of the microbial organism.