# US 2022/0033446 A1 · Systems and methods for discovering and optimizing lasso peptides

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- Number: US 2022/0033446 A1
- Document ID: US20220033446A1
- Status: Published application
- Kind: A1
- Application number: 17/296,372
- Filed: 2019-12-09
- Published: 2022-02-03
- Earliest claimed: 2018-12-10
- Assignees: Lassogen, Inc.
- Applicants: Lassogen, Inc.
- Inventors: Mark J. Burk; I-Hsiung Brandon Chen
- Web page: https://patentgrep.com/patent/US20220033446A1

## CPC

- C40B 40/10: Libraries containing peptides or polypeptides, or derivatives thereof
- C07K 7/56: the cyclisation not occurring through 2,4-diamino-butanoic acid
- C07K 14/245: Escherichia (G)
- C07K 14/36: from Actinomyces; from Streptomyces (G)
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/1075: {by coupling phenotype to genotype, not provided for in other groups of this subclass}
- C40B 30/00: Methods of screening libraries
- C02F 1/28: by sorption (using ion-exchange C02F1/42; sorbent compositions B01J)
- C02F 2103/30: from the textile industry
- C07K 2319/20: containing a tag with affinity for a non-protein ligand
- C07K 2319/21: containing a His-tag
- C07K 2319/50: containing protease site
- C07K 2319/70: containing domain for protein-protein interaction
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C02F: TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE (separation in general B01D; special arrangements on waterborne vessels of installations for treating water, waste water or sewage, e.g. for producing fresh water, B63J; adding materials to water to prevent corrosion C23F; treating radioactively-contaminated liquids G21F9/04; regeneration of reactants for recirculation into processes, see the relevant places for the processes)

## Abstract

Provided herein are lasso peptides libraries, and particularly molecular display libraries of lasso peptides. Also provided herein are related methods and systems for producing the libraries and for screening the libraries to identify candidate lasso peptides having desirable properties.

## Claims

### Claim 1

A lasso peptide display library comprising a plurality of members, wherein each member comprises a lasso peptide or a functional fragment of lasso peptide; and wherein each member is associated with a unique identification mechanism for distinguishing the plurality of members from one another, wherein the unique identification mechanism is a unique nucleic acid molecule or a unique location.

### Claim 2 (depends on 1)

The lasso peptide display library of claim 1, wherein the library further comprises a solid support.

### Claim 3 (depends on 2)

The lasso peptide display library of claim 2, wherein each member is associated with the unique identification mechanism through the solid support.

### Claim 4 (depends on 2)

The lasso peptide display library of claim 2, wherein the solid support comprises a plurality of unique locations, and each member is associated with one of the plurality of unique locations.

### Claim 5 (depends on 1, 2, 3, 4)

The lasso peptide display library of any one of claims 1-4, wherein at least one of the lasso peptide and/or functional fragment of lasso peptide forms part of a fusion protein.

### Claim 6 (depends on 1, 2, 3, 4, 5)

The lasso peptide display library of any one of claims 1-5, wherein at least one of the lasso peptide and/or functional fragment of lasso peptide forms part of a protein complex.

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The lasso peptide display library of any one of claims 1-6, wherein at least one of the lasso peptide and/or functional fragment of lasso peptide forms part of a conjugate.

### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7)

The lasso peptide display library of any one of claims 1-7, wherein the unique identification mechanism is a unique nucleic acid molecule.

### Claim 9 (depends on 8)

The lasso peptide display library of claim 8, wherein the lasso peptide or functional fragment of lasso peptide is fused to a first binding partner; and wherein the unique nucleic acid molecule is conjugated with a second binding partner.

### Claim 10 (depends on 9)

The lasso peptide display library of claim 9, wherein the first binding partner and the second binding partner are capable of directly or indirectly associating with one another.

### Claim 11 (depends on 9, 10)

The lasso peptide display library of claim 9 or 10, wherein the first binding partner and the second binding partner are both configured to associate with the solid support.

### Claim 12 (depends on 11)

The lasso peptide display library of claim 11, wherein the solid support is coated with or comprises a third binding partner capable of associating with the first binding partner and the second binding partner.

### Claim 13 (depends on 9, 10, 11, 12)

The lasso peptide display library of any one of claims 9-12, wherein the first binding partner is streptavidin; and wherein the second binding partner is biotin moiety conjugated with the unique nucleic acid molecule.

### Claim 14 (depends on 9, 10, 11, 12)

The lasso peptide display library of any one of claims 9-12, wherein the first binding partner is a nucleic acid binding protein and the second binding partner is target nucleic acid sequence that is a fragment of the unique nucleic acid molecule.

### Claim 15 (depends on 14)

The lasso peptide display library of claim 14, wherein the nucleic acid binding protein is replication protein RepA and the unique nucleic acid molecule comprises replication origin R (oriR) and cis-acting element (CIS) of RepA.

### Claim 16 (depends on 12)

The lasso peptide display library of claim 12, wherein the first binding partner is a streptavidin binding protein; wherein the second binding partner is biotin moiety conjugated with the unique nucleic acid molecule; and wherein the third binding partner is streptavidin.

### Claim 17 (depends on 9, 10, 11, 12, 13, 14, 15, 16)

The lasso peptide display library of any one of claims 9-16, wherein the solid support is a magnetic bead.

### Claim 18 (depends on 9, 10, 11, 12, 13, 14, 15, 16, 17)

The lasso peptide display library of any one of claims 9-17, wherein the lasso peptide or functional fragment thereof is associated with the unique nucleic acid molecule through a cleavable linker.

### Claim 19 (depends on 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The lasso peptide display library of any one of claims 8-18, wherein the unique nucleic acid molecule is a nucleic acid barcode.

### Claim 20 (depends on 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The lasso peptide display library of any one of claims 8-18, wherein the unique nucleic acid molecule encodes at least a portion of the lasso peptide or functional fragment thereof associated with the unique nucleic acid.

### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)

The lasso peptide display library of any one of claims 1-20, further comprising a cell-free biosynthesis system configured for providing the plurality of members.

### Claim 22 (depends on 21)

The lasso peptide display library of claim 21, wherein the cell-free biosynthesis system comprises a minimal set of lasso peptide biosynthesis components.

### Claim 23 (depends on 21, 22)

The lasso peptide display library of claim 21 or 22, wherein the minimal set of lasso peptide biosynthesis components comprises (i) at least one lasso precursor peptide or (ii) a first nucleic acid sequence encoding the at least one lasso precursor peptide and cell-free transcription-translation machinery.

### Claim 24 (depends on 21, 22, 23)

The lasso peptide display library of any one of claims 21-23, wherein the minimal set of lasso peptide biosynthesis components comprises (i) at least one lasso core peptide or (ii) a second nucleic acid sequence encoding the at least one lasso core peptide and cell-free transcription-translation machinery.

### Claim 25 (depends on 21, 22, 23, 24)

The lasso peptide display library of any one of claims 21-24, wherein the minimal set of lasso peptide biosynthesis components comprises (i) at least one lasso peptidase or (ii) a third nucleic acid sequence encoding the at least one lasso peptidase and cell-free transcription-translation machinery.

### Claim 26 (depends on 21, 22, 23, 24, 25)

The lasso peptide display library of any one of claims 21-25, wherein the minimal set of lasso peptide biosynthesis components comprises (i) at least one lasso cyclase or (ii) a fourth nucleic acid sequence encoding the at least one lasso cyclase and cell-free transcription-translation machinery.

### Claim 27 (depends on 21, 22, 23, 24, 25, 26)

The lasso peptide display library of any one of claims 21-26, wherein the minimal set of lasso peptide biosynthesis components comprises (i) at least one RiPP recognition element (RRE) or (ii) a fifth nucleic acid sequence encoding the at least one RRE and cell-free transcription-translation machinery.

### Claim 28 (depends on 21, 22, 23, 24, 25, 26, 27)

The lasso peptide display library of any one of claims 21-27, wherein the minimal set of lasso peptide biosynthesis components comprises
(i) a plurality of a first nucleic acid sequences each encoding a unique lasso precursor peptide;
(ii) at least one lasso peptidase or a third nucleic acid sequence encoding the lasso peptidase;
(iii) at least one lasso cyclase or a fourth nucleic acid sequence encoding the lasso cyclase; and
(iv) cell-free transcription-translation machinery.

### Claim 29 (depends on 28)

The lasso peptide display library of claim 28, wherein the plurality of the first nucleic acid sequences are derived from a same lasso peptide biosynthesis gene cluster.

### Claim 30 (depends on 29)

The lasso peptide display library of claim 29, wherein the plurality of the first nucleic acid sequences are obtained by randomly mutating Gene A of the same lasso peptide biosynthesis gene cluster.

### Claim 31 (depends on 29)

The lasso peptide display library of claim 29, wherein the random mutation is introduced to all codons of Gene A except for the ring-forming residue.

### Claim 32 (depends on 31)

The lasso peptide display library of claim 31, wherein the ring-forming residue is Glu at position 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or Asp at position 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

### Claim 33 (depends on 29)

The lasso peptide display library of claim 29, wherein the plurality of the first nucleic acid sequences are obtained by changing the position of the codon coding for the ring-forming residue in Gene A of the same lasso peptide biosynthesis gene cluster.

### Claim 34 (depends on 28)

The lasso peptide display library of claim 28, wherein the plurality of the first nucleic acid sequences are derived from a plurality of lasso peptide biosynthesis gene cluster.

### Claim 35 (depends on 28, 29, 30, 31, 32, 33, 34)

The lasso peptide display library of any one of claims 28-34, wherein the minimal set of lasso peptide biosynthesis components further comprises at least one RiPP recognition element (RRE) or a fifth nucleic acid sequence encoding the RRE.

### Claim 36 (depends on 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)

The lasso peptide display library of any one of claims 23-35, wherein at least one of the first, second, third, fourth and fifth nucleic acid sequences are operably linked to an expression control fragment.

### Claim 37 (depends on 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)

The lasso peptide display library of any one of claims 23-36, wherein at least two of the first, second, third, fourth and fifth nucleic acid sequences form part of a same nucleic acid molecule.

### Claim 38 (depends on 37)

The lasso peptide display library of claim 37, wherein at least two of the third, fourth and fifth nucleic acid sequences are fused in frame with each other in the same nucleic acid molecule.

### Claim 39 (depends on 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38)

The lasso peptide display library of any one of claims 23-38, wherein at least two of the first, second, third, fourth and fifth nucleic acids sequences comprise sequences derived from the same lasso peptide biosynthesis gene cluster.

### Claim 40 (depends on 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)

The lasso peptide display library of any one of claims 23-39, wherein at least two of the first, second, third, fourth and fifth nucleic acid sequences comprise sequences derived from different lasso peptide biosynthesis gene clusters.

### Claim 41 (depends on 40)

The lasso peptide display library of claim 40, wherein the third, fourth and fifth nucleic acid sequences comprise sequences derived from the same lasso peptide biosynthesis gene cluster of a host organism; and wherein the transcription-translation machinery is a cell lysate of the same host organism.

### Claim 42 (depends on 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)

The lasso peptide display library of any one of claims 23-35, wherein at least one of the first, second, third, fourth and fifth nucleic acid sequences is DNA, mRNA or cDNA sequence.

### Claim 43 (depends on 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)

The lasso peptide display library of any one of claims 23-42, wherein at least one of the first, second, third, fourth and fifth nucleic acid sequences further comprises a sequence encoding for a peptidic tag.

### Claim 44 (depends on 43)

The lasso peptide display library of claim 43, wherein the peptidic tag is a purification tag.

### Claim 45 (depends on 43)

The lasso peptide display library of claim 43, wherein the peptidic tag comprises a cleavable linker.

### Claim 46 (depends on 43)

The lasso peptide display library of claim 43, wherein the peptidic tag forms part of a binding partner.

### Claim 47 (depends on 43)

The lasso peptide display library of claim 43, wherein the peptidic tag produces a detectable signal.

### Claim 48 (depends on 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47)

The lasso peptide display library of any one of claims 21-47, wherein the cell-free biosynthesis system comprises cell lysate or supplemented cell lysate.

### Claim 49 (depends on 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)

The lasso peptide display library of any one of claims 21-48, wherein the cell-free biosynthesis system comprises components of cellular transcription-translation machinery purified from a cell.

### Claim 50 (depends on 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49)

The lasso peptide display library of any one of claims 21-49, wherein the cell-free biosynthesis system comprises synthetic or recombinantly produced components of cellular transcription-translation machinery.

### Claim 51 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50)

The lasso peptide display library of any one of claims 1 to 50, wherein the lasso peptide or a functional fragment of lasso peptide comprises at least one unnatural or unusual amino acid.

### Claim 52

A fusion protein comprising a lasso peptide component fused to a binding partner.

### Claim 53 (depends on 52)

The fusion protein according to claim 52, wherein the lasso peptide component is (i) a lasso peptide, (ii) a functional fragment of lasso peptide; (iii) a lasso precursor peptide; or (iv) a lasso core peptide.

### Claim 54 (depends on 52, 53)

The fusion protein according to claim 52 or 53, wherein the lasso peptide component is fused to the binding partner via a cleavable linker.

### Claim 55 (depends on 52, 53, 54)

The fusion protein according to any one of claims 52 to 54, wherein the binding partner is a streptavidin binding peptide (SBP), a streptavidin protein, or a nucleic acid binding protein.

### Claim 56 (depends on 55)

The fusion protein according to claim 55, wherein the nucleic acid binding protein is replication protein RepA.

### Claim 57 (depends on 52, 53, 54, 55, 56)

The fusion protein according to any one of claims 52 to 56, further comprising a purification tag.

### Claim 58 (depends on 57)

The fusion protein according to claim 57, wherein the purification tag is a His Tag.

### Claim 59 (depends on 52)

A nucleic acid molecule encoding the fusion protein according to any one of claim 52 to

### Claim 60 (depends on 59)

The nucleic acid molecule of claim 59, wherein the nucleic acid molecule is biotinylated.

### Claim 61 (depends on 59)

The nucleic acid molecule of claim 59, wherein the nucleic acid molecule further comprises the replication origin R (oriR) and cis-acting element (CIS) of RepA.

### Claim 62 (depends on 52, 53, 54, 55, 56, 57, 58)

A molecular complex comprising the fusion protein of any one of claims 52 to 58 and a nucleic acid molecule.

### Claim 63 (depends on 62)

The molecular complex according to claim 62, wherein the nucleic acid molecule encodes at least a portion of the lasso peptide fragment.

### Claim 64 (depends on 62)

The molecular complex according to claim 62, wherein the nucleic acid molecule is a unique member of a set of nucleic acid barcodes.

### Claim 65 (depends on 62, 63, 64)

The molecular complex according to any one of claims 62 to 64, wherein the nucleic acid molecule is biotinylated.

### Claim 66 (depends on 65)

The molecular complex according to claim 65, wherein the binding partner is the streptavidin protein.

### Claim 67 (depends on 65)

The molecular complex according to claim 65, wherein the binding partner is the streptavidin binding peptide (SBP), and wherein the molecular complex further comprises a streptavidin protein.

### Claim 68 (depends on 62, 63, 64)

The molecular complex according to any one of claims 62 to 64, wherein the nucleic acid molecule comprises the replication origin R (oriR) and cis-acting element (CIS) of RepA, and wherein the binding partner is RepA.

### Claim 69 (depends on 59, 60, 61, 62, 63, 64, 65, 66, 67, 68)

The molecular complex according to any one of claims 62 to 68, wherein the nucleic acid molecule is the nucleic acid molecule of any one of claims 59-61.

### Claim 70 (depends on 62, 63, 64, 65, 66)

A composition comprising a plurality of the molecular complexes according to any one of claims 62-66, wherein each of the plurality of the molecular complexes comprises a unique lasso peptide or functional fragment of lasso peptide.

### Claim 71

A method for evolving a lasso peptide of interest for a target property, the method comprising
a. providing a first lasso peptide display library comprising members derived from the lasso peptide of interest, wherein each member of the first lasso peptide display library comprises at least one mutation to the lasso peptide of interest;
b. subjecting the library to a first assay under a first condition to identify members having the target property;
c. identifying the mutations of the identified members as beneficial mutations; and
d. introducing the beneficial mutations into the lasso peptide of interest to provide an evolved lasso peptide.

### Claim 72 (depends on 71)

The method of claim 71, wherein the method further comprises:
f. providing an evolved lasso peptide display library comprising members derived from the evolved lasso peptide, wherein the members of the second library retain at least one beneficial mutation; and
g. repeating steps b through d.

### Claim 73 (depends on 72)

The method of claim 72, wherein the method further comprises repeating steps f and g for at least one more round.

### Claim 74 (depends on 71, 72, 73)

The method of any one of claims 71-73, wherein the evolved lasso peptide display library is subjected to the first assay under a second condition more stringent for the target property than the first condition.

### Claim 75 (depends on 72, 73, 74)

The method of any one of claims 72-74, wherein the evolved lasso peptide display library is subjected to a second assay to identify members having the target property.

### Claim 76 (depends on 71, 72, 73, 74, 75)

The method of any one of claims 71-75, wherein the method further comprises validating the evolved lasso peptide using at least one additional assay different from the first or second assay.

### Claim 77 (depends on 71, 72, 73, 74, 75, 76)

The method of any one of claims 71-76, wherein the target property is binding affinity for a target molecule.

### Claim 78 (depends on 71, 72, 73, 74, 75, 76)

The method of any one of claims 71-76, wherein the target property is binding specificity for a target molecule.

### Claim 79 (depends on 71, 72, 73, 74, 75, 76)

The method of any one of claims 71-76, wherein the target property is capability of modulating a cellular activity or cell phenotype.

### Claim 80 (depends on 78)

The method of claim 78, wherein the modulation is antagonist modulation or agonist modulation.

### Claim 81 (depends on 71, 72, 73, 74, 75, 76, 77, 78, 79, 80)

The method of any one of claims 71-80, wherein the mutation comprises substituting at least one amino acid with an unusual or unnatural amino acid.

### Claim 82 (depends on 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81)

The method of any one of claims 71 to 81, wherein the target property is at least two target properties screened simultaneously.

### Claim 83

A method for identifying a lasso peptide that specifically binds to a target molecule, the method comprising:
providing a lasso peptide display library comprising a plurality of members, each member comprising a lasso peptide or a functional fragment of lasso peptide;
contacting the library with the target molecule under a suitable condition that allows at least one member of the library to form a complex with the target molecule; and
identifying the member of in the complex.

### Claim 84 (depends on 82)

The method of claim 82,
wherein the contacting is performed by contacting the library with the target molecule in the presence of a reference binding partner of the target molecule under a suitable condition that allows at least one member of the library to compete with the reference binding partner for binding to the target molecule; and
wherein the identifying step is performed by detecting reduced binding of the reference binding partner to the target molecule; and identifying the member responsible for the reduced binding.

### Claim 85 (depends on 84)

The method of claim 84, wherein the reference binding partner is a ligand for the target molecule.

### Claim 86 (depends on 84, 85)

The method of claim 84 or 85, wherein the target molecule comprises one or more target sites, and the reference binding partner specifically binds to a target site of the target molecule.

### Claim 87 (depends on 85)

The method of claim 85, wherein the reference binding partner is a natural ligand or synthetic ligand for the target molecule.

### Claim 88 (depends on 83, 84, 85, 86, 87)

The method of any one of claims 83 to 87, wherein the target molecule is at least two target molecules.

### Claim 89

A method for identifying a lasso peptide that modulates a cellular activity, the method comprising
a. providing a lasso peptide display library comprising a plurality of members, each member comprising a lasso peptide or a functional fragment of lasso peptide;
b. subjecting the library to a suitable biological assay configured for measuring the cellular activity;
c. detecting a change in the cellular activity; and
d. identifying the members responsible for the detected change.

### Claim 90 (depends on 89)

The method of claim 89, wherein the step b is performed by subjecting the library to multiple biological assays configured for measuring the cellular activity; and the method further comprises selecting the members that have a high probability of being identified as responsible for the detected change in the cellular activity.

### Claim 91

A method for identifying an agonist or antagonist lasso peptide for a target molecule, the method comprising:
providing a lasso peptide display library comprising a plurality of members, each member comprising a lasso peptide or a functional fragment of lasso peptide;
contacting the library with a cell expressing the target molecule under a suitable condition that allows at least one member of the library to bind to the target molecule;
measuring a cellular activity mediated by the target molecule; and
identifying the member as an agonist ligand for the target molecule if said cellular activity is increased; or identifying the member as an antagonist ligand if said cellular activity is decreased.